LOCUS       sequence-346233-        3647 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  plasmid containing scaffold gRNA for Cas9 followed by a 28bp Csy4 
            recognition sequence, RPR1p-tetO-GFP-scRNA-28bpCsy4-RPR1t.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3647)
  AUTHORS   Otto M, Skrekas C, Gossing M, Gustafsson J, Siewers V, David F
  TITLE     Expansion of the Yeast Modular Cloning Toolkit for CRISPR-Based 
            Applications, Genomic Integrations and Combinatorial Libraries.
  JOURNAL   ACS Synth Biol. 2021 Dec 17;10(12):3461-3474. doi: 
            10.1021/acssynbio.1c00408. Epub 2021 Dec 3.
  PUBMED    34860007
REFERENCE   2  (bases 1 to 3647)
  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"; doi: 
            "10.1021/acssynbio.1c00408"; journalName: "ACS Synth Biol"; date: 
            "2021-12-17- 17"; volume: "10"; issue: "12"; pages: "3461-3474"
COMMENT     Sequence Label: pMCL8_28bp
FEATURES             Location/Qualifiers
     source          1..3647
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      193..781
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     682..701
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    1224..1242
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     RBS             1463..1474
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             1481..2194
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     terminator      2206..2277
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2293..2320
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     misc_RNA        2335..2410
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    2411..2430
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     terminator      2439..2501
                     /label=RPR1 terminator
                     /note="transcription terminator for the S. cerevisiae RNase
                     P RNA gene"
     terminator      complement(2894..2928)
                     /label=lambda t0 terminator
                     /note="minimal transcription terminator from phage lambda 
                     (Scholtissek and Grosse, 1987)"
     CDS             complement(join(2999..3647,1..11))
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     3573..3592
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
ORIGIN
        1 tttttctcca ttttagcttc cttagctcct gaaaatctcg ataactcaaa aaatacgccc
       61 ggtagtgatc ttatttcatt atggtgaaag ttggaacctc ttacgtgccc gatcaatcat
      121 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
      181 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
      241 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
      301 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt
      361 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
      421 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
      481 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
      541 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
      601 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
      661 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
      721 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
      781 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
      841 gttctttcct gcgttatccc ctgattctgt ggataaccgt agtcggtctc caacgggggg
      901 atctgccaat tgaacataac atggtagtta catatactag taatatggtt cggcacacat
      961 taaaagtata aaaactatct gaattacgaa ttacatatat tggtcataaa aatcaatcaa
     1021 tcatcgtgtg ttttatatgt ctcttatcta agtataagaa tatccatagt taatattcac
     1081 ttacgctacc ttttaacctg taatcattgt caacaggata tgttaacgac ccacattgat
     1141 aaacgctagt atttcttttt cctcttctta ttggccggct gtctctatac tcccctatag
     1201 tctgtttctt ttcgtttcga ttgtccctat cagtgataga gatggcgcac atggtacgct
     1261 gtggtgctcg cggctgggaa cgaaactctg ggagctgcga ttggcagtga gacggaaagt
     1321 gaaacgtgat ttcatgcgtc attttgaaca ttttgtaaat cttatttaat aatgtgtgcg
     1381 gcaattcaca tttaatttat gaatgttttc ttaacatcgc ggcaactcaa gaaacggcag
     1441 gttcggatct tagctactag agaaagagga gaaatactag atgcgtaaag gcgaagagct
     1501 gttcactggt gtcgtcccta ttctggtgga actggatggt gatgtcaacg gtcataagtt
     1561 ttccgtgcgt ggcgagggtg aaggtgacgc aactaatggt aaactgacgc tgaagttcat
     1621 ctgtactact ggtaaactgc cggttccttg gccgactctg gtaacgacgc tgacttatgg
     1681 tgttcagtgc tttgctcgtt atccggacca tatgaagcag catgacttct tcaagtccgc
     1741 catgccggaa ggctatgtgc aggaacgcac gatttccttt aaggatgacg gcacgtacaa
     1801 aacgcgtgcg gaagtgaaat ttgaaggcga taccctggta aaccgcattg agctgaaagg
     1861 cattgacttt aaagaggacg gcaatatcct gggccataag ctggaataca attttaacag
     1921 ccacaatgtt tacatcaccg ccgataaaca aaaaaatggc attaaagcga attttaaaat
     1981 tcgccacaac gtggaggatg gcagcgtgca gctggctgat cactaccagc aaaacactcc
     2041 aatcggtgat ggtcctgttc tgctgccaga caatcactat ctgagcacgc aaagcgttct
     2101 gtctaaagat ccgaacgaga aacgcgatca tatggttctg ctggagttcg taaccgcagc
     2161 gggcatcacg catggtatgg atgaactgta caaatgacca ggcatcaaat aaaacgaaag
     2221 gctcagtcga aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa cgctctctac
     2281 tagagtcaca ctggctcacc ttcgggtggg cctttctgcg tttatacgtc tcaggtttta
     2341 gagctagaaa tagcaagtta aaataaggct agtccgttat caacttgaaa aagtggcacc
     2401 gagtcggtgc gttcactgcc gtataggcag ctaagaaacc atatccaact tccaatttaa
     2461 tctttctttt ttaattttca cttatttgcg atacagaaag aaaaaagcga tagtaactat
     2521 tgaattttgt ttggatttgg ttagattaga tatggtttct ctttatattt acatgctaaa
     2581 aatgggctac accagagata cataattaga tatatatacg ccagtacacc ttatcggccc
     2641 aagccttgtc ccaaggcagc gttttgttct tggaaacgct gccctacacg ttcgctatgc
     2701 ttcaagaact tttctgagca cttcatgatg catgtttgtt ccttattggt tagctttgat
     2761 gttgtgaagt cattgacaca gtctgtgaaa catctttcta ccagattaga gtacaaacgc
     2821 atgaaatcct tcatttgctt ttgttccact actttttgga actcttgttg ttctttgctg
     2881 tgagaccaga ccaataaaaa acgcccggcg gcaaccgagc gttctgaaca aatccagatg
     2941 gagttctgag gtcattactg gatctatcaa caggagtcca agcgagctcg atatcaaatt
     3001 acgccccgcc ctgccactca tcgcagtact gttgtaattc attaagcatt ctgccgacat
     3061 ggaagccatc acaaacggca tgatgaacct gaatcgccag cggcatcagc accttgtcgc
     3121 cttgcgtata atatttgccc atggtgaaaa cgggggcgaa gaagttgtcc atattggcca
     3181 cgtttaaatc aaaactggtg aaactcaccc agggattggc tgaaacgaaa aacatattct
     3241 caataaaccc tttagggaaa taggccaggt tttcaccgta acacgccaca tcttgcgaat
     3301 atatgtgtag aaactgccgg aaatcgtcgt ggtattcact ccagagcgat gaaaacgttt
     3361 cagtttgctc atggaaaacg gtgtaacaag ggtgaacact atcccatatc accagctcac
     3421 cgtctttcat tgccatacga aattccggat gagcattcat caggcgggca agaatgtgaa
     3481 taaaggccgg ataaaacttg tgcttatttt tctttacggt ctttaaaaag gccgtaatat
     3541 ccagctgaac ggtctggtta taggtacatt gagcaactga ctgaaatgcc tcaaaatgtt
     3601 ctttacgatg ccattgggat atatcaacgg tggtatatcc agtgatt
//
