LOCUS       sequence-346497-        3626 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  gRNA cloning cassette with RPR1p-tetO promoter and terminator RPT1t,
            RPR1p-tetO-GFP-scRNA-RPR1t.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3626)
  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 3626)
  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
FEATURES             Location/Qualifiers
     source          1..3626
                     /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             1461..1472
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             1479..2192
                     /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      2204..2275
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2291..2318
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     misc_RNA        2331..2405
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      2418..2480
                     /label=RPR1 terminator
                     /note="transcription terminator for the S. cerevisiae RNase
                     P RNA gene"
     terminator      complement(2873..2907)
                     /label=lambda t0 terminator
                     /note="minimal transcription terminator from phage lambda 
                     (Scholtissek and Grosse, 1987)"
     CDS             complement(join(2978..3626,1..11))
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     3552..3571
                     /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 ttggcagaga cggaaagtga
     1321 aacgtgattt catgcgtcat tttgaacatt ttgtaaatct tatttaataa tgtgtgcggc
     1381 aattcacatt taatttatga atgttttctt aacatcgcgg caactcaaga aacggcaggt
     1441 tcggatctta gctactagag aaagaggaga aatactagat gcgtaaaggc gaagagctgt
     1501 tcactggtgt cgtccctatt ctggtggaac tggatggtga tgtcaacggt cataagtttt
     1561 ccgtgcgtgg cgagggtgaa ggtgacgcaa ctaatggtaa actgacgctg aagttcatct
     1621 gtactactgg taaactgccg gttccttggc cgactctggt aacgacgctg acttatggtg
     1681 ttcagtgctt tgctcgttat ccggaccata tgaagcagca tgacttcttc aagtccgcca
     1741 tgccggaagg ctatgtgcag gaacgcacga tttcctttaa ggatgacggc acgtacaaaa
     1801 cgcgtgcgga agtgaaattt gaaggcgata ccctggtaaa ccgcattgag ctgaaaggca
     1861 ttgactttaa agaggacggc aatatcctgg gccataagct ggaatacaat tttaacagcc
     1921 acaatgttta catcaccgcc gataaacaaa aaaatggcat taaagcgaat tttaaaattc
     1981 gccacaacgt ggaggatggc agcgtgcagc tggctgatca ctaccagcaa aacactccaa
     2041 tcggtgatgg tcctgttctg ctgccagaca atcactatct gagcacgcaa agcgttctgt
     2101 ctaaagatcc gaacgagaaa cgcgatcata tggttctgct ggagttcgta accgcagcgg
     2161 gcatcacgca tggtatggat gaactgtaca aatgaccagg catcaaataa aacgaaaggc
     2221 tcagtcgaaa gactgggcct ttcgttttat ctgttgtttg tcggtgaacg ctctctacta
     2281 gagtcacact ggctcacctt cgggtgggcc tttctgcgtt tatacgtctc ttttagagct
     2341 agaaatagca agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc
     2401 ggtgcttttt tctcgagcca tatccaactt ccaatttaat ctttcttttt taattttcac
     2461 ttatttgcga tacagaaaga aaaaagcgat agtaactatt gaattttgtt tggatttggt
     2521 tagattagat atggtttctc tttatattta catgctaaaa atgggctaca ccagagatac
     2581 ataattagat atatatacgc cagtacacct tatcggccca agccttgtcc caaggcagcg
     2641 ttttgttctt ggaaacgctg ccctacacgt tcgctatgct tcaagaactt ttctgagcac
     2701 ttcatgatgc atgtttgttc cttattggtt agctttgatg ttgtgaagtc attgacacag
     2761 tctgtgaaac atctttctac cagattagag tacaaacgca tgaaatcctt catttgcttt
     2821 tgttccacta ctttttggaa ctcttgttgt tctttgctgt gagaccagac caataaaaaa
     2881 cgcccggcgg caaccgagcg ttctgaacaa atccagatgg agttctgagg tcattactgg
     2941 atctatcaac aggagtccaa gcgagctcga tatcaaatta cgccccgccc tgccactcat
     3001 cgcagtactg ttgtaattca ttaagcattc tgccgacatg gaagccatca caaacggcat
     3061 gatgaacctg aatcgccagc ggcatcagca ccttgtcgcc ttgcgtataa tatttgccca
     3121 tggtgaaaac gggggcgaag aagttgtcca tattggccac gtttaaatca aaactggtga
     3181 aactcaccca gggattggct gaaacgaaaa acatattctc aataaaccct ttagggaaat
     3241 aggccaggtt ttcaccgtaa cacgccacat cttgcgaata tatgtgtaga aactgccgga
     3301 aatcgtcgtg gtattcactc cagagcgatg aaaacgtttc agtttgctca tggaaaacgg
     3361 tgtaacaagg gtgaacacta tcccatatca ccagctcacc gtctttcatt gccatacgaa
     3421 attccggatg agcattcatc aggcgggcaa gaatgtgaat aaaggccgga taaaacttgt
     3481 gcttattttt ctttacggtc tttaaaaagg ccgtaatatc cagctgaacg gtctggttat
     3541 aggtacattg agcaactgac tgaaatgcct caaaatgttc tttacgatgc cattgggata
     3601 tatcaacggt ggtatatcca gtgatt
//
