LOCUS       sequence-252914-        2868 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  classic sgRNA backbone for SpCas9, combine with AtU6-26 promoter 
            module (pFH35).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2868)
  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 2868)
  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: pFH99
FEATURES             Location/Qualifiers
     source          1..2868
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    76..97
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(112..117,118..135,136..142)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    150..166
                     /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)."
     primer_bind     174..190
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             186..509
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHACRSTLEDPRVPSSNSLAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICS
                     DAA"
     misc_feature    203..259
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(260..276)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(485..504)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     misc_RNA        607..682
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(760..776)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     rep_origin      complement(948..1536)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1028..1047)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(1672..2487)
                     /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             complement(1672..2487)
                     /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     2395..2414
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2479..2498
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     2608..2624
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2608..2624
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     terminator      2625..2671
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     2800..2816
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 gggtctcaat tgttgtcttc gcagctggca cgacaggttt cccgactgga aagcgggcag
       61 tgagcgcaac gcaattaatg tgagttagct cactcattag gcaccccagg ctttacactt
      121 tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa
      181 cagctatgac catgattacg ccaagcttgc atgcctgcag gtcgactcta gaggatcccc
      241 gggtaccgag ctcgaattca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg
      301 gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg
      361 aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatggcgcc
      421 tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata tggtgcactc
      481 tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg
      541 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgacga
      601 agacaagttt tagagctaga aatagcaagt taaaataagg ctagtccgtt atcaacttga
      661 aaaagtggca ccgagtcggt gctttttttt tcgcttgaga ccgaggctag gtggaggctc
      721 agtgatgata agtctgcgat ggtggatgca tgtgtcatgg tcatagctgt ttcctgtgtg
      781 aaattgttat ccgctcagag ggcacaatcc tattccgcgc tatccgacaa tctccaagac
      841 attaggtgga gttcagttcg gcgtatggca tatgtcgctg gaaagaacat gtgagcaaaa
      901 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
      961 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     1021 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     1081 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     1141 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     1201 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     1261 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     1321 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     1381 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     1441 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     1501 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     1561 gggtctgacg ctctattcaa caaagccgcc gtcccgtcaa gtcagcgtaa atgggtaggg
     1621 ggcttcaaat cgtccgctct gccagtgtta caaccaatta acaaattctg attagaaaaa
     1681 ctcatcgagc atcaaatgaa actgcaattt attcatatca ggattatcaa taccatattt
     1741 ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg aggcagttcc ataggatggc
     1801 aagatcctgg tatcggtctg cgattccgac tcgtccaaca tcaatacaac ctattaattt
     1861 cccctcgtca aaaataaggt tatcaagtga gaaatcacca tgagtgacga ctgaatccgg
     1921 tgagaatggc aaaagcttat gcatttcttt ccagacttgt tcaacaggcc agccattacg
     1981 ctcgtcatca aaatcactcg catcaaccaa accgttattc attcgtgatt gcgcctgagc
     2041 gagacgaaat acgcgatcgc tgttaaaagg acaattacaa acaggaatcg aatgcaaccg
     2101 gcgcaggaac actgccagcg catcaacaat attttcacct gaatcaggat attcttctaa
     2161 tacctggaat gctgttttcc cggggatcgc agtggtgagt aaccatgcat catcaggagt
     2221 acggataaaa tgcttgatgg tcggaagagg cataaattcc gtcagccagt ttagtctgac
     2281 catctcatct gtaacatcat tggcaacgct acctttgcca tgtttcagaa acaactctgg
     2341 cgcatcgggc ttcccataca atcgatagat tgtcgcacct gattgcccga cattatcgcg
     2401 agcccattta tacccatata aatcagcatc catgttggaa tttaatcgcg gcctcgagca
     2461 agacgtttcc cgttgaatat ggctcataac accccttgta ttactgttta tgtaagcaga
     2521 cagttttatt gttcatgatg atatattttt atcttgtgca atgtaacatc agagattttg
     2581 agacacaacg tggctttccc ccgccgctct agaactagtg gatccaaata aaacgaaagg
     2641 ctcagtcgaa agactgggcc tttcgtttta tctgttgttt gtcgcattat acgagacgtc
     2701 caggttggga tacctgaaac aaaacccatc gtacggccaa ggaagtctcc aataactgtg
     2761 atccaccaca agcgccaggg ttttcccagt cacgacgttg taaaacgacg gccagtcatg
     2821 cataatccgc acgcatctgg aataaggaag tgccattccg cctgacct
//
