LOCUS       sequence-249038-        2764 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Guide-Generating Vector for creating the Cys4-guide-scaffold 
            structure in the first round of assembly.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2764)
  AUTHORS   McCarty NS, Shaw WM, Ellis T, Ledesma-Amaro R
  TITLE     Rapid Assembly of gRNA Arrays via Modular Cloning in Yeast.
  JOURNAL   ACS Synth Biol. 2019 Apr 2. doi: 10.1021/acssynbio.9b00041.
  PUBMED    30939239
REFERENCE   2  (bases 1 to 2764)
  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. 2019 Apr 2. doi: 10.1021/acssynbio.9b00041."
COMMENT     Sequence Label: Guide-Generating Vector
FEATURES             Location/Qualifiers
     source          1..2764
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    1..20
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     RBS             176..187
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             194..907
                     /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      919..990
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1006..1033
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     misc_RNA        1047..1122
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      complement(1131..1165)
                     /label=lambda t0 terminator
                     /note="minimal transcription terminator from phage lambda 
                     (Scholtissek and Grosse, 1987)"
     CDS             complement(1236..1895)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     1810..1829
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     rep_origin      2077..2665
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2566..2585
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gttcactgcc gtataggcag tgagacggaa agtgaaacgt gatttcatgc gtcattttga
       61 acattttgta aatcttattt aataatgtgt gcggcaattc acatttaatt tatgaatgtt
      121 ttcttaacat cgcggcaact caagaaacgg caggttcgga tcttagctac tagagaaaga
      181 ggagaaatac tagatgcgta aaggcgaaga gctgttcact ggtgtcgtcc ctattctggt
      241 ggaactggat ggtgatgtca acggtcataa gttttccgtg cgtggcgagg gtgaaggtga
      301 cgcaactaat ggtaaactga cgctgaagtt catctgtact actggtaaac tgccggttcc
      361 ttggccgact ctggtaacga cgctgactta tggtgttcag tgctttgctc gttatccgga
      421 ccatatgaag cagcatgact tcttcaagtc cgccatgccg gaaggctatg tgcaggaacg
      481 cacgatttcc tttaaggatg acggcacgta caaaacgcgt gcggaagtga aatttgaagg
      541 cgataccctg gtaaaccgca ttgagctgaa aggcattgac tttaaagagg acggcaatat
      601 cctgggccat aagctggaat acaattttaa cagccacaat gtttacatca ccgccgataa
      661 acaaaaaaat ggcattaaag cgaattttaa aattcgccac aacgtggagg atggcagcgt
      721 gcagctggct gatcactacc agcaaaacac tccaatcggt gatggtcctg ttctgctgcc
      781 agacaatcac tatctgagca cgcaaagcgt tctgtctaaa gatccgaacg agaaacgcga
      841 tcatatggtt ctgctggagt tcgtaaccgc agcgggcatc acgcatggta tggatgaact
      901 gtacaaatga ccaggcatca aataaaacga aaggctcagt cgaaagactg ggcctttcgt
      961 tttatctgtt gtttgtcggt gaacgctctc tactagagtc acactggctc accttcgggt
     1021 gggcctttct gcgtttatac gtctcagttt tagagctaga aatagcaagt taaaataagg
     1081 ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gcttttacca ataaaaaacg
     1141 cccggcggca accgagcgtt ctgaacaaat ccagatggag ttctgaggtc attactggat
     1201 ctatcaacag gagtccaagc gagctcgata tcaaattacg ccccgccctg ccactcatcg
     1261 cagtactgtt gtaattcatt aagcattctg ccgacatgga agccatcaca aacggcatga
     1321 tgaacctgaa tcgccagcgg catcagcacc ttgtcgcctt gcgtataata tttgcccatg
     1381 gtgaaaacgg gggcgaagaa gttgtccata ttggccacgt ttaaatcaaa actggtgaaa
     1441 ctcacccagg gattggctga aacgaaaaac atattctcaa taaacccttt agggaaatag
     1501 gccaggtttt caccgtaaca cgccacatct tgcgaatata tgtgtagaaa ctgccggaaa
     1561 tcgtcgtggt attcactcca gagcgatgaa aacgtttcag tttgctcatg gaaaacggtg
     1621 taacaagggt gaacactatc ccatatcacc agctcaccgt ctttcattgc catacgaaat
     1681 tccggatgag cattcatcag gcgggcaaga atgtgaataa aggccggata aaacttgtgc
     1741 ttatttttct ttacggtctt taaaaaggcc gtaatatcca gctgaacggt ctggttatag
     1801 gtacattgag caactgactg aaatgcctca aaatgttctt tacgatgcca ttgggatata
     1861 tcaacggtgg tatatccagt gatttttttc tccattttag cttccttagc tcctgaaaat
     1921 ctcgataact caaaaaatac gcccggtagt gatcttattt cattatggtg aaagttggaa
     1981 cctcttacgt gcccgatcaa tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     2041 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     2101 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     2161 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     2221 tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     2281 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     2341 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     2401 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     2461 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     2521 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     2581 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     2641 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     2701 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa
     2761 ccgt
//
