LOCUS       sequence-252926-        2878 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  improved sgRNA backbone for SpCas9, combine with TaU3 promoter 
            module (pFH33).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2878)
  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 2878)
  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: pFH86
FEATURES             Location/Qualifiers
     source          1..2878
                     /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"
     primer_bind     complement(770..786)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     rep_origin      complement(958..1546)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1038..1057)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(1682..2497)
                     /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(1682..2497)
                     /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     2405..2424
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2489..2508
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     2618..2634
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2618..2634
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     terminator      2635..2681
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     2810..2826
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 gggtctcaag cattgtcttc 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 cagagctatg ctggaaacag catagcaagt tgaaataagg ctagtccgtt
      661 atcaacttga aaaagtggca ccgagtcggt gctttttttt tcgcttgaga ccgaggctag
      721 gtggaggctc agtgatgata agtctgcgat ggtggatgca tgtgtcatgg tcatagctgt
      781 ttcctgtgtg aaattgttat ccgctcagag ggcacaatcc tattccgcgc tatccgacaa
      841 tctccaagac attaggtgga gttcagttcg gcgtatggca tatgtcgctg gaaagaacat
      901 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
      961 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     1021 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     1081 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     1141 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     1201 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     1261 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     1321 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     1381 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     1441 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     1501 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     1561 cttttctacg gggtctgacg ctctattcaa caaagccgcc gtcccgtcaa gtcagcgtaa
     1621 atgggtaggg ggcttcaaat cgtccgctct gccagtgtta caaccaatta acaaattctg
     1681 attagaaaaa ctcatcgagc atcaaatgaa actgcaattt attcatatca ggattatcaa
     1741 taccatattt ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg aggcagttcc
     1801 ataggatggc aagatcctgg tatcggtctg cgattccgac tcgtccaaca tcaatacaac
     1861 ctattaattt cccctcgtca aaaataaggt tatcaagtga gaaatcacca tgagtgacga
     1921 ctgaatccgg tgagaatggc aaaagcttat gcatttcttt ccagacttgt tcaacaggcc
     1981 agccattacg ctcgtcatca aaatcactcg catcaaccaa accgttattc attcgtgatt
     2041 gcgcctgagc gagacgaaat acgcgatcgc tgttaaaagg acaattacaa acaggaatcg
     2101 aatgcaaccg gcgcaggaac actgccagcg catcaacaat attttcacct gaatcaggat
     2161 attcttctaa tacctggaat gctgttttcc cggggatcgc agtggtgagt aaccatgcat
     2221 catcaggagt acggataaaa tgcttgatgg tcggaagagg cataaattcc gtcagccagt
     2281 ttagtctgac catctcatct gtaacatcat tggcaacgct acctttgcca tgtttcagaa
     2341 acaactctgg cgcatcgggc ttcccataca atcgatagat tgtcgcacct gattgcccga
     2401 cattatcgcg agcccattta tacccatata aatcagcatc catgttggaa tttaatcgcg
     2461 gcctcgagca agacgtttcc cgttgaatat ggctcataac accccttgta ttactgttta
     2521 tgtaagcaga cagttttatt gttcatgatg atatattttt atcttgtgca atgtaacatc
     2581 agagattttg agacacaacg tggctttccc ccgccgctct agaactagtg gatccaaata
     2641 aaacgaaagg ctcagtcgaa agactgggcc tttcgtttta tctgttgttt gtcgcattat
     2701 acgagacgtc caggttggga tacctgaaac aaaacccatc gtacggccaa ggaagtctcc
     2761 aataactgtg atccaccaca agcgccaggg ttttcccagt cacgacgttg taaaacgacg
     2821 gccagtcatg cataatccgc acgcatctgg aataaggaag tgccattccg cctgacct
//
