LOCUS       Exported                7664 bp ds-DNA     circular SYN 12-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
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
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"
ORIGIN
        1 ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag
       61 ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa
      121 accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga
      181 ctggaaagcc gcggatgatt acgccaagcg cgcaattaac caatacgcaa accgcctctc
      241 cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcc
      301 gcggatgatt acgccaagcg cgcaattaac cctcactaaa gggaacaaaa gctggagctc
      361 gggggatctg ccaattgaac ataacatggt agttacatat actagtaata tggttcggca
      421 cacattaaaa gtataaaaac tatctgaatt acgaattaca tatattggtc ataaaaatca
      481 atcaatcatc gtgtgtttta tatgtctctt atctaagtat aagaatatcc atagttaata
      541 ttcacttacg ctacctttta acctgtaatc attgtcaaca ggatatgtta acgacccaca
      601 ttgataaacg ctagtatttc tttttcctct tcttattggc cggctgtctc tatactcccc
      661 tatagtctgt ttcttttcgt ttcgattgtt ttacgtttga ggcctcgtgg cgcacatggt
      721 acgctgtggt gctcgcggct gggaacgaaa ctctgggagc tgcgattggc agaagcttgt
      781 tttagagcta gaaatagcaa gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg
      841 caccgagtcg gtgctttttt ctcgagccat atccaacttc caatttaatc tttctttttt
      901 aattttcact tatttgcgat acagaaagaa aaaagcgata gtaactattg aattttgttt
      961 ggatttggtt agattagata tggtttctct ttatatttac atgctaaaaa tgggctacac
     1021 cagagataca taattagata tatatacgcc agtacacctt atcggcccaa gccttgtccc
     1081 aaggcagcgt tttgttcttg gaaacgctgc cctacacgtt cgctatgctt caagaacttt
     1141 tctgagcact tcatgatgca tgtttgttcc ttattggtta gctttgatgt tgtgaagtca
     1201 ttgacacagt ctgtgaaaca tctttctacc agattagagt acaaacgcat gaaatccttc
     1261 atttgctttt gttccactac tttttggaac tcttgttgtt ctttggtacc caattcgccc
     1321 tatagtgagt cgtattacgc gcgctcactg gccgtcgttt tacaacgtcg tgactgggaa
     1381 aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt
     1441 aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa
     1501 tggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     1561 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
     1621 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     1681 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
     1741 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     1801 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
     1861 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
     1921 ttaacgcgaa ttttaacaaa atattaacgc ttacaatttc ctgatgcggt attttctcct
     1981 tacgcatctg tgcggtattt cacaccgcat atcgaccggt cgaggagaac ttctagtata
     2041 tctacatacc taatattatt gccttattaa aaatggaatc ccaacaatta catcaaaatc
     2101 cacattctct tcaaaatcaa ttgtcctgta cttccttgtt catgtgtgtt caaaaacgtt
     2161 atatttatag gataattata ctctatttct caacaagtaa ttggttgttt ggccgagcgg
     2221 tctaaggcgc ctgattcaag aaatatcttg accgcagtta actgtgggaa tactcaggta
     2281 tcgtaagatg caagagttcg aatctcttag caaccattat ttttttcctc aacataacga
     2341 gaacacacag gggcgctatc gcacagaatc aaattcgatg actggaaatt ttttgttaat
     2401 ttcagaggtc gcctgacgca tatacctttt tcaactgaaa aattgggaga aaaaggaaag
     2461 gtgagagcgc cggaaccggc ttttcatata gaatagagaa gcgttcatga ctaaatgctt
     2521 gcatcacaat acttgaagtt gacaatatta tttaaggacc tattgttttt tccaataggt
     2581 ggttagcaat cgtcttactt tctaactttt cttacctttt acatttcagc aatatatata
     2641 tatatatttc aaggatatac cattctaatg tctgccccta agaagatcgt cgttttgcca
     2701 ggtgaccacg ttggtcaaga aatcacagcc gaagccatta aggttcttaa agctatttct
     2761 gatgttcgtt ccaatgtcaa gttcgatttc gaaaatcatt taattggtgg tgctgctatc
     2821 gatgctacag gtgttccact tccagatgag gcgctggaag cctccaagaa ggctgatgcc
     2881 gttttgttag gtgctgtggg tggtcctaaa tggggtaccg gtagtgttag acctgaacaa
     2941 ggtttactaa aaatccgtaa agaacttcaa ttgtacgcca acttaagacc atgtaacttt
     3001 gcatccgact ctcttttaga cttatctcca atcaagccac aatttgctaa aggtactgac
     3061 ttcgttgttg tcagagaatt agtgggaggt atttactttg gtaagagaaa ggaagacgat
     3121 ggtgatggtg tcgcttggga tagtgaacaa tacaccgttc cagaagtgca aagaatcaca
     3181 agaatggccg ctttcatggc cctacaacat gagccaccat tgcctatttg gtccttggat
     3241 aaagctaatg ttttggcctc ttcaagatta tggagaaaaa ctgtggagga aaccatcaag
     3301 aacgaattcc ctacattgaa ggttcaacat caattgattg attctgccgc catgatccta
     3361 gttaagaacc caacccacct aaatggtatt ataatcacca gcaacatgtt tggtgatatc
     3421 atctccgatg aagcctccgt tatcccaggt tccttgggtt tgttgccatc tgcgtccttg
     3481 gcctctttgc cagacaagaa caccgcattt ggtttgtacg aaccatgcca cggttctgct
     3541 ccagatttgc caaagaataa ggtcaaccct atcgccacta tcttgtctgc tgcaatgatg
     3601 ttgaaattgt cattgaactt gcctgaagaa ggtaaggcca ttgaagatgc agttaaaaag
     3661 gttttggatg caggtatcag aactggtgat ttaggtggtt ccaacagtac caccgaagtc
     3721 ggtgatgctg tcgccgaaga agttaagaaa atccttgctt aaaaagattc tcttttttta
     3781 tgatatttgt acataaactt tataaatgaa attcataata gaaacgacac gaaattacaa
     3841 aatggaatat gttcataggg tagacgaaac tatatacgca atctacatac atttatcaag
     3901 aaggagaaaa aggaggatgt aaaggaatac aggtaagcaa attgatacta atggctcaac
     3961 gtgataagga aaaagaattg cactttaaca ttaatattga caaggaggag ggcaccacac
     4021 aaaaagttag gtgtaacaga aaatcatgaa actatgattc ctaatttata tattggagga
     4081 ttttctctaa aaaaaaaaaa atacaacaaa taaaaaacac tcaatgacct gaccatttga
     4141 tggagtttaa gtcaatacct tcttgaacca tttcccataa tggtgaaagt tccctcaaga
     4201 attttactct gtcagaaacg gccttaacga cgtagtcgat atggtgcact ctcagtacaa
     4261 tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc gctgacgcgc
     4321 cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga
     4381 gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgagacga aagggcctcg
     4441 tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag tatgatccaa
     4501 tatcaaagga aatgatagca ttgaaggatg agactaatcc aattgaggag tggcagcata
     4561 tagaacagct aaagggtagt gctgaaggaa gcatacgata ccccgcatgg aatgggataa
     4621 tatcacagga ggtactagac tacctttcat cctacataaa tagacgcata taagtacgca
     4681 tttaagcata aacacgcact atgccgttct tctcatgtat atatatatac aggcaacacg
     4741 cagatatagg tgcgacgtga acagtgagct gtatgtgcgc agctcgcgtt gcattttcgg
     4801 aagcgctcgt tttcggaaac gctttgaagt tcctattccg aagttcctat tctctagaaa
     4861 gtataggaac ttcagagcgc ttttgaaaac caaaagcgct ctgaagacgc actttcaaaa
     4921 aaccaaaaac gcaccggact gtaacgagct actaaaatat tgcgaatacc gcttccacaa
     4981 acattgctca aaagtatctc tttgctatat atctctgtgc tatatcccta tataacctac
     5041 ccatccacct ttcgctcctt gaacttgcat ctaaactcga cctctacatt ttttatgttt
     5101 atctctagta ttactcttta gacaaaaaaa ttgtagtaag aactattcat agagtgaatc
     5161 gaaaacaata cgaaaatgta aacatttcct atacgtagta tatagagaca aaatagaaga
     5221 aaccgttcat aattttctga ccaatgaaga atcatcaacg ctatcacttt ctgttcacaa
     5281 agtatgcgca atccacatcg gtatagaata taatcgggga tgcctttatc ttgaaaaaat
     5341 gcacccgcag cttcgctagt aatcagtaaa cgcgggaagt ggagtcaggc tttttttatg
     5401 gaagagaaaa tagacaccaa agtagccttc ttctaacctt aacggaccta cagtgcaaaa
     5461 agttatcaag agactgcatt atagagcgca caaaggagaa aaaaagtaat ctaagatgct
     5521 ttgttagaaa aatagcgctc tcgggatgca tttttgtaga acaaaaaaga agtatagatt
     5581 ctttgttggt aaaatagcgc tctcgcgttg catttctgtt ctgtaaaaat gcagctcaga
     5641 ttctttgttt gaaaaattag cgctctcgcg ttgcattttt gttttacaaa aatgaagcac
     5701 agattcttcg ttggtaaaat agcgctttcg cgttgcattt ctgttctgta aaaatgcagc
     5761 tcagattctt tgtttgaaaa attagcgctc tcgcgttgca tttttgttct acaaaatgaa
     5821 gcacagatgc ttcgttcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     5881 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
     5941 cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt
     6001 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta
     6061 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc
     6121 ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa
     6181 gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc
     6241 cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt
     6301 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact
     6361 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac
     6421 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata
     6481 ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta
     6541 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg
     6601 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat
     6661 aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt
     6721 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga
     6781 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa
     6841 gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
     6901 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     6961 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     7021 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     7081 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     7141 actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     7201 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     7261 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     7321 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     7381 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     7441 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     7501 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     7561 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     7621 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatc
//
