LOCUS       sequence-367562-        2616 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for amplification of gRNA with Cas6 recognition site.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2616)
  AUTHORS   Volke DC, Martino RA, Kozaeva E, Smania AM, Nikel PI
  TITLE     Modular (de)construction of complex bacterial phenotypes by 
            CRISPR/nCas9-assisted, multiplex cytidine base-editing.
  JOURNAL   Nat Commun. 2022 May 31;13(1):3026. doi: 10.1038/s41467-022-30780-z.
  PUBMED    35641501
REFERENCE   2  (bases 1 to 2616)
  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: "2022-05-31"; volume: "13(1):3026. doi"; pages: " 
            10.1038/s41467-022-30780-z"
COMMENT     Sequence Label: pEX128·gRNA
FEATURES             Location/Qualifiers
     source          1..2616
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_RNA        32..107
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    108..127
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     protein_bind    207..223
                     /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)."
     promoter        complement(join(231..237,238..255,256..261))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    276..297
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(414..431)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(585..1173)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(665..684)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1344..2135,2136..2204))
                     /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     1967..1986
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2205..2309)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2377..2395
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2433..2455)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2555..2574
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 ggtctcagtg gcgacggcca gaagatcggt tgttttagag ctagaaatag caagttaaaa
       61 taaggctagt ccgttatcaa cttgaaaaag tggcaccgag tcggtgcgtt cactgccgta
      121 taggcagcta agaaaggcca accagatcaa gccgagttta gagaccacct gcttttgctc
      181 gcttggatcc gaattcaaag gtgaaattgt tatccgctca caattccaca caacatacga
      241 gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt
      301 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
      361 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc
      421 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
      481 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
      541 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
      601 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
      661 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
      721 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
      781 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
      841 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
      901 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
      961 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     1021 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     1081 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     1141 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     1201 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     1261 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     1321 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     1381 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     1441 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgactccc acgctcaccg
     1501 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     1561 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     1621 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
     1681 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     1741 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     1801 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     1861 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     1921 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
     1981 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     2041 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     2101 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     2161 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     2221 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     2281 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc
     2341 taagaaacca ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt
     2401 cgtctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct cccggagacg
     2461 gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg
     2521 ggtgttggcg ggtgtcgggg ctggcttaac tatgcggcat cagagcagat tgtactgaga
     2581 gaaaggcaat tgggtaccga gctcgcggcc gcaagc
//
