pRPR1_gRNA_handle_RPR1t

LOCUS       pRPR1_gRNA_handle_RPR1t                7693 bp ds-DNA     circular SYN 10-JAN-2018
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 7693)
  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
COMMENT     This file is created by Vector NTI
            http://www.biofeng.com/
COMMENT     ORIGDB|GenBank
COMMENT     VNTAUTHORNAME|biofeng.com|
COMMENT     VNTNAME|pRPR1_gRNA_handle_RPR1t|
FEATURES             Location/Qualifiers
     source          1..7693
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     complement(10..28)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     rep_origin      69..1411
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    401..448
                     /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        1438..1542
                     /gene="bla"
                     /label=AmpR promoter
     CDS             1543..2403
                     /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(1761..1780)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2574..3162
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3063..3082
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3316..3333
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        3486..3516
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3524..3540
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     3529..3551
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3548..3564
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3548..3564
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     3583..3603
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        3585..3603
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        3626..4032
                     /gene="RPR1"
                     /label=RPR1 promoter
                     /note="promoter for the S. cerevisiae RNase P RNA gene"
     misc_RNA        4040..4115
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      4128..4190
                     /label=RPR1 terminator
                     /note="transcription terminator for the S. cerevisiae RNase
                     P RNA gene"
     primer_bind     complement(4579..4598)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4580..4598)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4608..4625)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4608..4624)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4617..4639)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4765..5220
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4852..4871)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5062..5083
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5520..5924
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     CDS             5937..7031
                     /codon_start=1
                     /gene="S. cerevisiae LEU2"
                     /product="3-isopropylmalate dehydrogenase, required for 
                     leucine biosynthesis"
                     /label=LEU2
                     /note="yeast auxotrophic marker"
                     /translation="MSAPKKIVVLPGDHVGQEITAEAIKVLKAISDVRSNVKFDFENHL
                     IGGAAIDATGVPLPDEALEASKKVDAVLLGAVAGPKWGTGSVRPEQGLLKIRKELQLYA
                     NLRPCNFASDSLLDLSPIKPQFAKGTDFVVVRELVGGIYFGKRKEDDGDGVAWDSEQYT
                     VPEVQRITRMAAFMALQHEPPLPIWSLDKANLLASSRLWRKTVEETIKNEFPTLKVQHQ
                     LIDSAAMILVKNPTHLNGIIITSNMFGDIISDEASVIPGSLGLLPSASLASLPDKNTAF
                     GLYEPCHGSAPDLPKNKVDPIATILSAAMMLKLSLNLPEEGKAIEDAVKKVLDAGIRTG
                     DLGGSNSTTEVGDAVAEEVKKILA"
     primer_bind     complement(7524..7543)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7643..7665
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
       61 cttagtatga tccaatatca aaggaaatga tagcattgaa ggatgagact aatccaattg
      121 aggagtggca gcatatagaa cagctaaagg gtagtgctga aggaagcata cgataccccg
      181 catggaatgg gataatatca caggaggtac tagactacct ttcatcctac ataaatagac
      241 gcatataagt acgcatttaa gcataaacac gcactatgcc gttcttctca tgtatatata
      301 tatacaggca acacgcagat ataggtgcga cgtgaacagt gagctgtatg tgcgcagctc
      361 gcgttgcatt ttcggaagcg ctcgttttcg gaaacgcttt gaagttccta ttccgaagtt
      421 cctattctct agaaagtata ggaacttcag agcgcttttg aaaaccaaaa gcgctctgaa
      481 gacgcacttt caaaaaacca aaaacgcacc ggactgtaac gagctactaa aatattgcga
      541 ataccgcttc cacaaacatt gctcaaaagt atctctttgc tatatatctc tgtgctatat
      601 ccctatataa cctacccatc cacctttcgc tccttgaact tgcatctaaa ctcgacctct
      661 acatttttta tgtttatctc tagtattact ctttagacaa aaaaattgta gtaagaacta
      721 ttcatagagt gaatcgaaaa caatacgaaa atgtaaacat ttcctatacg tagtatatag
      781 agacaaaata gaagaaaccg ttcataattt tctgaccaat gaagaatcat caacgctatc
      841 actttctgtt cacaaagtat gcgcaatcca catcggtata gaatataatc ggggatgcct
      901 ttatcttgaa aaaatgcacc cgcagcttcg ctagtaatca gtaaacgcgg gaagtggagt
      961 caggcttttt ttatggaaga gaaaatagac accaaagtag ccttcttcta accttaacgg
     1021 acctacagtg caaaaagtta tcaagagact gcattataga gcgcacaaag gagaaaaaaa
     1081 gtaatctaag atgctttgtt agaaaaatag cgctctcggg atgcattttt gtagaacaaa
     1141 aaagaagtat agattctttg ttggtaaaat agcgctctcg cgttgcattt ctgttctgta
     1201 aaaatgcagc tcagattctt tgtttgaaaa attagcgctc tcgcgttgca tttttgtttt
     1261 acaaaaatga agcacagatt cttcgttggt aaaatagcgc tttcgcgttg catttctgtt
     1321 ctgtaaaaat gcagctcaga ttctttgttt gaaaaattag cgctctcgcg ttgcattttt
     1381 gttctacaaa atgaagcaca gatgcttcgt tcaggtggca cttttcgggg aaatgtgcgc
     1441 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
     1501 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc
     1561 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa
     1621 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa
     1681 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg
     1741 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa
     1801 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc
     1861 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc
     1921 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta
     1981 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag
     2041 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca
     2101 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata
     2161 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
     2221 tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca
     2281 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
     2341 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
     2401 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa
     2461 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     2521 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     2581 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     2641 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     2701 gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac
     2761 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     2821 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     2881 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     2941 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     3001 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     3061 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     3121 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     3181 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
     3241 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
     3301 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa
     3361 ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac
     3421 tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt acctcactca ttaggcaccc
     3481 caggctttac actttatgct tccggctcct atgttgtgtg gaattgtgag cggataacaa
     3541 tttcacacag gaaacagcta tgaccatgat tacgccaagc gcgcaattaa ccctcactaa
     3601 agggaacaaa agctggagct cgggggatct gccaattgaa cataacatgg tagttacata
     3661 tactagtaat atggttcggc acacattaaa agtataaaaa ctatctgaat tacgaattac
     3721 atatattggt cataaaaatc aatcaatcat cgtgtgtttt atatgtctct tatctaagta
     3781 taagaatatc catagttaat attcacttac gctacctttt aacctgtaat cattgtcaac
     3841 aggatatgtt aacgacccac attgataaac gctagtattt ctttttcctc ttcttattgg
     3901 ccggctgtct ctatactccc ctatagtctg tttcttttcg tttcgattgt tttacgtttg
     3961 aggcctcgtg gcgcacatgg tacgctgtgg tgctcgcggc tgggaacgaa actctgggag
     4021 ctgcgattgg cagaagcttg ttttagagct agaaatagca agttaaaata aggctagtcc
     4081 gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt tctcgagcca tatccaactt
     4141 ccaatttaat ctttcttttt taattttcac ttatttgcga tacagaaaga aaaaagcgat
     4201 agtaactatt gaattttgtt tggatttggt tagattagat atggtttctc tttatattta
     4261 catgctaaaa atgggctaca ccagagatac ataattagat atatatacgc cagtacacct
     4321 tatcggccca agccttgtcc caaggcagcg ttttgttctt ggaaacgctg ccctacacgt
     4381 tcgctatgct tcaagaactt ttctgagcac ttcatgatgc atgtttgttc cttattggtt
     4441 agctttgatg ttgtgaagtc attgacacag tctgtgaaac atctttctac cagattagag
     4501 tacaaacgca tgaaatcctt catttgcttt tgttccacta ctttttggaa ctcttgttgt
     4561 tctttggtac ccaattcgcc ctatagtgag tcgtattacg cgcgctcact ggccgtcgtt
     4621 ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat
     4681 ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag
     4741 ttgcgcagcc tgaatggcga atggacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     4801 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
     4861 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     4921 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     4981 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     5041 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta
     5101 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa
     5161 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg tttacaattt
     5221 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatcgacggt
     5281 cgaggagaac ttctagtata tccacatacc taatattatt gccttattaa aaatggaatc
     5341 ccaacaatta catcaaaatc cacattctct tcaaaatcaa ttgtcctgta cttccttgtt
     5401 catgtgtgtt caaaaacgtt atatttatag gataattata ctctatttct caacaagtaa
     5461 ttggttgttt ggccgagcgg tctaaggcgc ctgattcaag aaatatcttg accgcagtta
     5521 actgtgggaa tactcaggta tcgtaagatg caagagttcg aatctcttag caaccattat
     5581 ttttttcctc aacataacga gaacacacag gggcgctatc gcacagaatc aaattcgatg
     5641 actggaaatt ttttgttaat ttcagaggtc gcctgacgca tatacctttt tcaactgaaa
     5701 aattgggaga aaaaggaaag gtgagaggcc ggaaccggct tttcatatag aatagagaag
     5761 cgttcatgac taaatgcttg catcacaata cttgaagttg acaatattat ttaaggacct
     5821 attgtttttt ccaataggtg gttagcaatc gtcttacttt ctaacttttc ttacctttta
     5881 catttcagca atatatatat atatttcaag gatataccat tctaatgtct gcccctatgt
     5941 ctgcccctaa gaagatcgtc gttttgccag gtgaccacgt tggtcaagaa atcacagccg
     6001 aagccattaa ggttcttaaa gctatttctg atgttcgttc caatgtcaag ttcgatttcg
     6061 aaaatcattt aattggtggt gctgctatcg atgctacagg tgtcccactt ccagatgagg
     6121 cgctggaagc ctccaagaag gttgatgccg ttttgttagg tgctgtggct ggtcctaaat
     6181 ggggtaccgg tagtgttaga cctgaacaag gtttactaaa aatccgtaaa gaacttcaat
     6241 tgtacgccaa cttaagacca tgtaactttg catccgactc tcttttagac ttatctccaa
     6301 tcaagccaca atttgctaaa ggtactgact tcgttgttgt cagagaatta gtgggaggta
     6361 tttactttgg taagagaaag gaagacgatg gtgatggtgt cgcttgggat agtgaacaat
     6421 acaccgttcc agaagtgcaa agaatcacaa gaatggccgc tttcatggcc ctacaacatg
     6481 agccaccatt gcctatttgg tccttggata aagctaatct tttggcctct tcaagattat
     6541 ggagaaaaac tgtggaggaa accatcaaga acgaattccc tacattgaag gttcaacatc
     6601 aattgattga ttctgccgcc atgatcctag ttaagaaccc aacccaccta aatggtatta
     6661 taatcaccag caacatgttt ggtgatatca tctccgatga agcctccgtt atcccaggtt
     6721 ccttgggttt gttgccatct gcgtccttgg cctctttgcc agacaagaac accgcatttg
     6781 gtttgtacga accatgccac ggttctgctc cagatttgcc aaagaataag gttgacccta
     6841 tcgccactat cttgtctgct gcaatgatgt tgaaattgtc attgaacttg cctgaagaag
     6901 gtaaggccat tgaagatgca gttaaaaagg ttttggatgc aggtatcaga actggtgatt
     6961 taggtggttc caacagtacc accgaagtcg gtgatgctgt cgccgaagaa gttaagaaaa
     7021 tccttgctta aaaagattct ctttttttat gatatttgta cataaacttt ataaatgaaa
     7081 ttcataatag aaacgacacg aaattacaaa atggaatatg ttcatagggt agacgaaact
     7141 atatacgcaa tctacataca tttatcaaga aggagaaaaa ggaggatagt aaaggaatac
     7201 aggtaagcaa attgatacta atggctcaac gtgataagga aaaagaattg cactttaaca
     7261 ttaatattga caaggaggag ggcaccacac aaaaagttag gtgtaacaga aaatcatgaa
     7321 actacgattc ctaatttgat attggaggat tttctctaaa aaaaaaaaaa tacaacaaat
     7381 aaaaaacact caatgacctg accatttgat ggagtttaag tcaatacctt cttgaagcat
     7441 ttcccataat ggtgaaagtt ccctcaagaa ttttactctg tcagaaacgg ccttacgacg
     7501 tagtcgatat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc
     7561 cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct
     7621 tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca
     7681 ccgaaacgcg cga
//