LOCUS       sequence-104049-        7713 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  aTc inducible gRNA expression vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7713)
  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 7713)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "ACS Synth 
            Biol. 2013 Sep 11."
COMMENT     Sequence Label: pRPR1(1xTetO)_gRNA_handle_RPR1t
FEATURES             Location/Qualifiers
     source          1..7713
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     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             join(1543..1611,1612..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        join(3486..3491,3492..3509,3510..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"
     protein_bind    3950..3968
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     misc_RNA        4060..4135
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      4148..4210
                     /label=RPR1 terminator
                     /note="transcription terminator for the S. cerevisiae RNase
                     P RNA gene"
     primer_bind     complement(4599..4618)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4600..4618)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4628..4645)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4628..4644)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4637..4659)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4785..5240
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4872..4891)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5082..5103
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5540..5944
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     CDS             5957..7051
                     /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(7544..7563)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7663..7685
                     /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 ccctatcagt
     3961 gatagagatg gcgcacatgg tacgctgtgg tgctcgcggc tgggaacgaa actctgggag
     4021 ctgcgattgg cagaagcttt tactgtatgt acatacagtg ttttagagct agaaatagca
     4081 agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt
     4141 tctcgagcca tatccaactt ccaatttaat ctttcttttt taattttcac ttatttgcga
     4201 tacagaaaga aaaaagcgat agtaactatt gaattttgtt tggatttggt tagattagat
     4261 atggtttctc tttatattta catgctaaaa atgggctaca ccagagatac ataattagat
     4321 atatatacgc cagtacacct tatcggccca agccttgtcc caaggcagcg ttttgttctt
     4381 ggaaacgctg ccctacacgt tcgctatgct tcaagaactt ttctgagcac ttcatgatgc
     4441 atgtttgttc cttattggtt agctttgatg ttgtgaagtc attgacacag tctgtgaaac
     4501 atctttctac cagattagag tacaaacgca tgaaatcctt catttgcttt tgttccacta
     4561 ctttttggaa ctcttgttgt tctttggtac ccaattcgcc ctatagtgag tcgtattacg
     4621 cgcgctcact ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac
     4681 ttaatcgcct tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca
     4741 ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga atggacgcgc cctgtagcgg
     4801 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     4861 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     4921 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     4981 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac
     5041 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     5101 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat
     5161 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa
     5221 aatattaacg tttacaattt cctgatgcgg tattttctcc ttacgcatct gtgcggtatt
     5281 tcacaccgca tatcgacggt cgaggagaac ttctagtata tccacatacc taatattatt
     5341 gccttattaa aaatggaatc ccaacaatta catcaaaatc cacattctct tcaaaatcaa
     5401 ttgtcctgta cttccttgtt catgtgtgtt caaaaacgtt atatttatag gataattata
     5461 ctctatttct caacaagtaa ttggttgttt ggccgagcgg tctaaggcgc ctgattcaag
     5521 aaatatcttg accgcagtta actgtgggaa tactcaggta tcgtaagatg caagagttcg
     5581 aatctcttag caaccattat ttttttcctc aacataacga gaacacacag gggcgctatc
     5641 gcacagaatc aaattcgatg actggaaatt ttttgttaat ttcagaggtc gcctgacgca
     5701 tatacctttt tcaactgaaa aattgggaga aaaaggaaag gtgagaggcc ggaaccggct
     5761 tttcatatag aatagagaag cgttcatgac taaatgcttg catcacaata cttgaagttg
     5821 acaatattat ttaaggacct attgtttttt ccaataggtg gttagcaatc gtcttacttt
     5881 ctaacttttc ttacctttta catttcagca atatatatat atatttcaag gatataccat
     5941 tctaatgtct gcccctatgt ctgcccctaa gaagatcgtc gttttgccag gtgaccacgt
     6001 tggtcaagaa atcacagccg aagccattaa ggttcttaaa gctatttctg atgttcgttc
     6061 caatgtcaag ttcgatttcg aaaatcattt aattggtggt gctgctatcg atgctacagg
     6121 tgtcccactt ccagatgagg cgctggaagc ctccaagaag gttgatgccg ttttgttagg
     6181 tgctgtggct ggtcctaaat ggggtaccgg tagtgttaga cctgaacaag gtttactaaa
     6241 aatccgtaaa gaacttcaat tgtacgccaa cttaagacca tgtaactttg catccgactc
     6301 tcttttagac ttatctccaa tcaagccaca atttgctaaa ggtactgact tcgttgttgt
     6361 cagagaatta gtgggaggta tttactttgg taagagaaag gaagacgatg gtgatggtgt
     6421 cgcttgggat agtgaacaat acaccgttcc agaagtgcaa agaatcacaa gaatggccgc
     6481 tttcatggcc ctacaacatg agccaccatt gcctatttgg tccttggata aagctaatct
     6541 tttggcctct tcaagattat ggagaaaaac tgtggaggaa accatcaaga acgaattccc
     6601 tacattgaag gttcaacatc aattgattga ttctgccgcc atgatcctag ttaagaaccc
     6661 aacccaccta aatggtatta taatcaccag caacatgttt ggtgatatca tctccgatga
     6721 agcctccgtt atcccaggtt ccttgggttt gttgccatct gcgtccttgg cctctttgcc
     6781 agacaagaac accgcatttg gtttgtacga accatgccac ggttctgctc cagatttgcc
     6841 aaagaataag gttgacccta tcgccactat cttgtctgct gcaatgatgt tgaaattgtc
     6901 attgaacttg cctgaagaag gtaaggccat tgaagatgca gttaaaaagg ttttggatgc
     6961 aggtatcaga actggtgatt taggtggttc caacagtacc accgaagtcg gtgatgctgt
     7021 cgccgaagaa gttaagaaaa tccttgctta aaaagattct ctttttttat gatatttgta
     7081 cataaacttt ataaatgaaa ttcataatag aaacgacacg aaattacaaa atggaatatg
     7141 ttcatagggt agacgaaact atatacgcaa tctacataca tttatcaaga aggagaaaaa
     7201 ggaggatagt aaaggaatac aggtaagcaa attgatacta atggctcaac gtgataagga
     7261 aaaagaattg cactttaaca ttaatattga caaggaggag ggcaccacac aaaaagttag
     7321 gtgtaacaga aaatcatgaa actacgattc ctaatttgat attggaggat tttctctaaa
     7381 aaaaaaaaaa tacaacaaat aaaaaacact caatgacctg accatttgat ggagtttaag
     7441 tcaatacctt cttgaagcat ttcccataat ggtgaaagtt ccctcaagaa ttttactctg
     7501 tcagaaacgg ccttacgacg tagtcgatat ggtgcactct cagtacaatc tgctctgatg
     7561 ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt
     7621 gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc
     7681 agaggttttc accgtcatca ccgaaacgcg cga
//
