LOCUS       sequence-271528-        2812 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  crRNA backbone for FnCas12a/CmsI nucleases, combine with OsU3 
            promoter module (pFH39).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2812)
  AUTHORS   Hahn F, Korolev A, Sanjurjo Loures L, Nekrasov V
  TITLE     A modular cloning toolkit for genome editing in plants.
  JOURNAL   BMC Plant Biol. 2020 Apr 23;20(1):179. doi: 
            10.1186/s12870-020-02388-2.
  PUBMED    32326901
REFERENCE   2  (bases 1 to 2812)
  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: "BMC Plant 
            Biol."; date: "2020-04-23"; volume: "20(1):179. doi"; pages: " 
            10.1186/s12870-020-02388-2"
COMMENT     Sequence Label: pFH131
FEATURES             Location/Qualifiers
     source          1..2812
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     108..124
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             complement(299..622)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHACRSTLEDPRVPSSNSLAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICS
                     DAA"
     primer_bind     304..323
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     532..548
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    549..605
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(618..634)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    642..658
                     /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(666..672,673..690,691..696))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    711..732
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(880..896)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     terminator      complement(1025..1071)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(1072..1088)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1072..1088)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(1198..1217)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1209..2024
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             1209..2024
                     /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="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(1282..1301)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      2160..2748
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2649..2668
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ttccagcgac atatgccata cgccgaactg aactccacct aatgtcttgg agattgtcgg
       61 atagcgcgga ataggattgt gccctctgag cggataacaa tttcacacag gaaacagcta
      121 tgaccatgac acatgcatcc accatcgcag acttatcatc actgagcctc cacctagcct
      181 cggtctcaag cgaaaaaaaa attgtcttcg tcacagcttg tctgtaagcg gatgccggga
      241 gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc tggcttaact
      301 atgcggcatc agagcagatt gtactgagag tgcaccatat gcggtgtgaa ataccgcaca
      361 gatgcgtaag gagaaaatac cgcatcaggc gccattcgcc attcaggctg cgcaactgtt
      421 gggaagggcg atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa gggggatgtg
      481 ctgcaaggcg attaagttgg gtaacgccag ggttttccca gtcacgacgt tgtaaaacga
      541 cggccagtga attcgagctc ggtacccggg gatcctctag agtcgacctg caggcatgca
      601 agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt
      661 ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc
      721 taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
      781 cagctgcgaa gacaaatcta caacagtaga aattatgcct gagacccagg tcaggcggaa
      841 tggcacttcc ttattccaga tgcgtgcgga ttatgcatga ctggccgtcg ttttacaacg
      901 tcgtgactgg gaaaaccctg gcgcttgtgg tggatcacag ttattggaga cttccttggc
      961 cgtacgatgg gttttgtttc aggtatccca acctggacgt ctcgtataat gcgacaaaca
     1021 acagataaaa cgaaaggccc agtctttcga ctgagccttt cgttttattt ggatccacta
     1081 gttctagagc ggcgggggaa agccacgttg tgtctcaaaa tctctgatgt tacattgcac
     1141 aagataaaaa tatatcatca tgaacaataa aactgtctgc ttacataaac agtaatacaa
     1201 ggggtgttat gagccatatt caacgggaaa cgtcttgctc gaggccgcga ttaaattcca
     1261 acatggatgc tgatttatat gggtataaat gggctcgcga taatgtcggg caatcaggtg
     1321 cgacaatcta tcgattgtat gggaagcccg atgcgccaga gttgtttctg aaacatggca
     1381 aaggtagcgt tgccaatgat gttacagatg agatggtcag actaaactgg ctgacggaat
     1441 ttatgcctct tccgaccatc aagcatttta tccgtactcc tgatgatgca tggttactca
     1501 ccactgcgat ccccgggaaa acagcattcc aggtattaga agaatatcct gattcaggtg
     1561 aaaatattgt tgatgcgctg gcagtgttcc tgcgccggtt gcattcgatt cctgtttgta
     1621 attgtccttt taacagcgat cgcgtatttc gtctcgctca ggcgcaatca cgaatgaata
     1681 acggtttggt tgatgcgagt gattttgatg acgagcgtaa tggctggcct gttgaacaag
     1741 tctggaaaga aatgcataag cttttgccat tctcaccgga ttcagtcgtc actcatggtg
     1801 atttctcact tgataacctt atttttgacg aggggaaatt aataggttgt attgatgttg
     1861 gacgagtcgg aatcgcagac cgataccagg atcttgccat cctatggaac tgcctcggtg
     1921 agttttctcc ttcattacag aaacggcttt ttcaaaaata tggtattgat aatcctgata
     1981 tgaataaatt gcagtttcat ttgatgctcg atgagttttt ctaatcagaa tttgttaatt
     2041 ggttgtaaca ctggcagagc ggacgatttg aagcccccta cccatttacg ctgacttgac
     2101 gggacggcgg ctttgttgaa tagagcgtca gaccccgtag aaaagatcaa aggatcttct
     2161 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     2221 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     2281 agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc
     2341 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     2401 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     2461 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     2521 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     2581 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     2641 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     2701 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     2761 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ct
//
