LOCUS       sequence-393062-        3701 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid for cloning of a CRISPR-Cas9 guide RNA gene under promoter 
            PJ23119.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3701)
  AUTHORS   Li C, Wang L, Cseke LJ, Vasconcelos F, Huguet-Tapia JC, Gassmann W, 
            Pauwels L, White FF, Dong H, Yang B
  TITLE     Efficient CRISPR-Cas9 based cytosine base editors for 
            phytopathogenic bacteria.
  JOURNAL   Commun Biol. 2023 Jan 17;6(1):56. doi: 10.1038/s42003-023-04451-8.
  PUBMED    36646768
REFERENCE   2  (bases 1 to 3701)
  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: "Commun 
            Biol."; date: "2023-01-17"; volume: "6(1):56. doi"; pages: " 
            10.1038/s42003-023-04451-8"
COMMENT     Sequence Label: pEN-L1-PJ23119-PplpT-sfGFP-TrrfB-BsaI-Scaf-L2
FEATURES             Location/Qualifiers
     source          1..3701
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     77..94
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(253..280)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(372..458)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     507..529
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     521..538
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     522..538
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    554..653
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        660..694
                     /label=J23119 promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119)"
     RBS             850..861
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             868..1581
                     /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      1593..1664
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1680..1707
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     misc_RNA        1723..1798
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(1811..1910)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1927..1946)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1928..1946)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1951..1967)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1951..1967)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2069..2088)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2080..2889
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(2147..2166)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      3036..3624
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3525..3544
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag
       61 ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa
      121 accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga
      181 ctggaaagcg ggcagtgagc gcaacgcaat taatacgcgt accgctagcc aggaagagtt
      241 tgtagaaacg caaaaaggcc atccgtcagg atggccttct gcttagtttg atgcctggca
      301 gtttatggcg ggcgtcctgc ccgccaccct ccgggccgtt gcttcacaac gttcaaatcc
      361 gctcccggcg gatttgtcct actcaggaga gcgttcaccg acaaacaaca gataaaacga
      421 aaggcccagt cttccgactg agcctttcgt tttatttgat gcctggcagt tccctactct
      481 cgcgttaacg ctagcatgga tgttttccca gtcacgacgt tgtaaaacga cggccagtct
      541 taagctcggg ccccaaataa tgattttatt ttgactgata gtgacctgtt cgttgcaaca
      601 aattgatgag caatgctttt ttataatgcc aactttgtac aaaaaagcag gcttaacctt
      661 tgacagctag ctcagtccta ggtataatgc tagcggagac cgaaagtgaa acgtgatttc
      721 atgcgtcatt ttgaacattt tgtaaatctt atttaataat gtgtgcggca attcacattt
      781 aatttatgaa tgttttctta acatcgcggc aactcaagaa acggcaggtt cggatcttag
      841 ctactagaga aagaggagaa atactagatg cgtaaaggcg aagagctgtt cactggtgtc
      901 gtccctattc tggtggaact ggatggtgat gtcaacggtc ataagttttc cgtgcgtggc
      961 gagggtgaag gtgacgcaac taatggtaaa ctgacgctga agttcatctg tactactggt
     1021 aaactgccgg ttccttggcc gactctggta acgacgctga cttatggtgt tcagtgcttt
     1081 gctcgttatc cggaccatat gaagcagcat gacttcttca agtccgccat gccggaaggc
     1141 tatgtgcagg aacgcacgat ttcctttaag gatgacggca cgtacaaaac gcgtgcggaa
     1201 gtgaaatttg aaggcgatac cctggtaaac cgcattgagc tgaaaggcat tgactttaaa
     1261 gaggacggca atatcctggg ccataagctg gaatacaatt ttaacagcca caatgtttac
     1321 atcaccgccg ataaacaaaa aaatggcatt aaagcgaatt ttaaaattcg ccacaacgtg
     1381 gaggatggca gcgtgcagct ggctgatcac taccagcaaa acactccaat cggtgatggt
     1441 cctgttctgc tgccagacaa tcactatctg agcacgcaaa gcgttctgtc taaagatccg
     1501 aacgagaaac gcgatcatat ggttctgctg gagttcgtaa ccgcagcggg catcacgcat
     1561 ggtatggatg aactgtacaa atgaccaggc atcaaataaa acgaaaggct cagtcgaaag
     1621 actgggcctt tcgttttatc tgttgtttgt cggtgaacgc tctctactag agtcacactg
     1681 gctcaccttc gggtgggcct ttctgcgttt atagaggtct ctgttttaga gctagaaata
     1741 gcaagttaaa ataaggctag tccgttatca acttgaaaaa gtggcaccga gtcggtgctt
     1801 tttttgtatt acccagcttt cttgtacaaa gttggcatta taagaaagca ttgcttatca
     1861 atttgttgca acgaacaggt cactatcagt caaaataaaa tcattatttg ccatccagct
     1921 gatatcccct atagtgagtc gtattacatg gtcatagctg tttcctggca gctctggccc
     1981 gtgtctcaaa atctctgatg ttacattgca caagataaaa atatatcatc atgaacaata
     2041 aaactgtctg cttacataaa cagtaataca aggggtgtta tgagccatat tcaacgggaa
     2101 acgtcgaggc cgcgattaaa ttccaacatg gatgctgatt tatatgggta taaatgggct
     2161 cgcgataatg tcgggcaatc aggtgcgaca atctatcgct tgtatgggaa gcccgatgcg
     2221 ccagagttgt ttctgaaaca tggcaaaggt agcgttgcca atgatgttac agatgagatg
     2281 gtcagactaa actggctgac ggaatttatg cctcttccga ccatcaagca ttttatccgt
     2341 actcctgatg atgcatggtt actcaccact gcgatccccg gaaaaacagc attccaggta
     2401 ttagaagaat atcctgattc aggtgaaaat attgttgatg cgctggcagt gttcctgcgc
     2461 cggttgcatt cgattcctgt ttgtaattgt ccttttaaca gcgatcgcgt atttcgtctc
     2521 gctcaggcgc aatcacgaat gaataacggt ttggttgatg cgagtgattt tgatgacgag
     2581 cgtaatggct ggcctgttga acaagtctgg aaagaaatgc ataaactttt gccattctca
     2641 ccggattcag tcgtcactca tggtgatttc tcacttgata accttatttt tgacgagggg
     2701 aaattaatag gttgtattga tgttggacga gtcggaatcg cagaccgata ccaggatctt
     2761 gccatcctat ggaactgcct cggtgagttt tctccttcat tacagaaacg gctttttcaa
     2821 aaatatggta ttgataatcc tgatatgaat aaattgcagt ttcatttgat gctcgatgag
     2881 tttttctaat cagaattggt taattggttg taacactggc agagcattac gctgacttga
     2941 cgggacggcg caagctcatg accaaaatcc cttaacgtga gttacgcgtc gttccactga
     3001 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     3061 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     3121 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     3181 gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     3241 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     3301 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     3361 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     3421 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     3481 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     3541 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     3601 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     3661 ttttgctggc cttttgctca catgttcttt cctgcgttat c
//
