LOCUS       sequence-358355-        1750 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  SpCas9 gRNA-Csy4 template.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 1750)
  AUTHORS   Shaw WM, Studena L, Roy K, Hapeta P, McCarty NS, Graham AE, Ellis T,
            Ledesma-Amaro R
  TITLE     Inducible expression of large gRNA arrays for multiplexed CRISPRai 
            applications.
  JOURNAL   Nat Commun. 2022 Aug 25;13(1):4984. doi: 10.1038/s41467-022-32603-7.
  PUBMED    36008396
REFERENCE   2  (bases 1 to 1750)
  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: "Nat 
            Commun."; date: "2022-08-25"; volume: "13(1):4984. doi"; pages: " 
            10.1038/s41467-022-32603-7"
COMMENT     Sequence Label: pWS3799-gRNA-Csy4-template
FEATURES             Location/Qualifiers
     source          1..1750
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_RNA        7..82
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    87..106
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     terminator      complement(117..151)
                     /label=lambda t0 terminator
                     /note="minimal transcription terminator from phage lambda 
                     (Scholtissek and Grosse, 1987)"
     CDS             complement(222..881)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     796..815
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     rep_origin      1063..1651
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1552..1571
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 agtcgggttt tagagctaga aatagcaagt taaaataagg ctagtccgtt atcaacttga
       61 aaaagtggca ccgagtcggt gcttttgttc actgccgtat aggcaggacc agaccaataa
      121 aaaacgcccg gcggcaaccg agcgttctga acaaatccag atggagttct gaggtcatta
      181 ctggatctat caacaggagt ccaagcgagc tcgatatcaa attacgcccc gccctgccac
      241 tcatcgcagt actgttgtaa ttcattaagc attctgccga catggaagcc atcacaaacg
      301 gcatgatgaa cctgaatcgc cagcggcatc agcaccttgt cgccttgcgt ataatatttg
      361 cccatggtga aaacgggggc gaagaagttg tccatattgg ccacgtttaa atcaaaactg
      421 gtgaaactca cccagggatt ggctgaaacg aaaaacatat tctcaataaa ccctttaggg
      481 aaataggcca ggttttcacc gtaacacgcc acatcttgcg aatatatgtg tagaaactgc
      541 cggaaatcgt cgtggtattc actccagagc gatgaaaacg tttcagtttg ctcatggaaa
      601 acggtgtaac aagggtgaac actatcccat atcaccagct caccgtcttt cattgccata
      661 cgaaattccg gatgagcatt catcaggcgg gcaagaatgt gaataaaggc cggataaaac
      721 ttgtgcttat ttttctttac ggtctttaaa aaggccgtaa tatccagctg aacggtctgg
      781 ttataggtac attgagcaac tgactgaaat gcctcaaaat gttctttacg atgccattgg
      841 gatatatcaa cggtggtata tccagtgatt tttttctcca ttttagcttc cttagctcct
      901 gaaaatctcg ataactcaaa aaatacgccc ggtagtgatc ttatttcatt atggtgaaag
      961 ttggaacctc ttacgtgccc gatcaatcat gaccaaaatc ccttaacgtg agttttcgtt
     1021 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     1081 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     1141 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     1201 aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     1261 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     1321 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     1381 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     1441 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     1501 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     1561 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     1621 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     1681 cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt
     1741 ggataaccgt
//
