LOCUS       sequence-252918-        2813 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  crRNA backbone for LbCas12a, combine with TaU3 promoter module 
            (pFH33).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2813)
  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 2813)
  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: pFH89
FEATURES             Location/Qualifiers
     source          1..2813
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    97..118
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(133..138,139..156,157..163)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    171..187
                     /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     195..211
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             207..530
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHACRSTLEDPRVPSSNSLAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICS
                     DAA"
     misc_feature    224..280
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(281..297)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(506..525)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(705..721)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     rep_origin      complement(893..1481)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(973..992)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(1617..2432)
                     /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(1617..2432)
                     /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     2340..2359
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2424..2443
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     2553..2569
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2553..2569
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     terminator      2570..2616
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     2745..2761
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 gggtctcaag cataatttct actaagtgta gatttgtctt cgcagctggc acgacaggtt
       61 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta
      121 ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg
      181 ataacaattt cacacaggaa acagctatga ccatgattac gccaagcttg catgcctgca
      241 ggtcgactct agaggatccc cgggtaccga gctcgaattc actggccgtc gttttacaac
      301 gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt
      361 tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca
      421 gcctgaatgg cgaatggcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt
      481 cacaccgcat atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagc
      541 cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg
      601 cttacagaca agctgtgacg aagacaattt ttttttcgct tgagaccgag gctaggtgga
      661 ggctcagtga tgataagtct gcgatggtgg atgcatgtgt catggtcata gctgtttcct
      721 gtgtgaaatt gttatccgct cagagggcac aatcctattc cgcgctatcc gacaatctcc
      781 aagacattag gtggagttca gttcggcgta tggcatatgt cgctggaaag aacatgtgag
      841 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
      901 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
      961 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     1021 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     1081 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     1141 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     1201 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     1261 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     1321 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     1381 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     1441 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     1501 ctacggggtc tgacgctcta ttcaacaaag ccgccgtccc gtcaagtcag cgtaaatggg
     1561 tagggggctt caaatcgtcc gctctgccag tgttacaacc aattaacaaa ttctgattag
     1621 aaaaactcat cgagcatcaa atgaaactgc aatttattca tatcaggatt atcaatacca
     1681 tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact caccgaggca gttccatagg
     1741 atggcaagat cctggtatcg gtctgcgatt ccgactcgtc caacatcaat acaacctatt
     1801 aatttcccct cgtcaaaaat aaggttatca agtgagaaat caccatgagt gacgactgaa
     1861 tccggtgaga atggcaaaag cttatgcatt tctttccaga cttgttcaac aggccagcca
     1921 ttacgctcgt catcaaaatc actcgcatca accaaaccgt tattcattcg tgattgcgcc
     1981 tgagcgagac gaaatacgcg atcgctgtta aaaggacaat tacaaacagg aatcgaatgc
     2041 aaccggcgca ggaacactgc cagcgcatca acaatatttt cacctgaatc aggatattct
     2101 tctaatacct ggaatgctgt tttcccgggg atcgcagtgg tgagtaacca tgcatcatca
     2161 ggagtacgga taaaatgctt gatggtcgga agaggcataa attccgtcag ccagtttagt
     2221 ctgaccatct catctgtaac atcattggca acgctacctt tgccatgttt cagaaacaac
     2281 tctggcgcat cgggcttccc atacaatcga tagattgtcg cacctgattg cccgacatta
     2341 tcgcgagccc atttataccc atataaatca gcatccatgt tggaatttaa tcgcggcctc
     2401 gagcaagacg tttcccgttg aatatggctc ataacacccc ttgtattact gtttatgtaa
     2461 gcagacagtt ttattgttca tgatgatata tttttatctt gtgcaatgta acatcagaga
     2521 ttttgagaca caacgtggct ttcccccgcc gctctagaac tagtggatcc aaataaaacg
     2581 aaaggctcag tcgaaagact gggcctttcg ttttatctgt tgtttgtcgc attatacgag
     2641 acgtccaggt tgggatacct gaaacaaaac ccatcgtacg gccaaggaag tctccaataa
     2701 ctgtgatcca ccacaagcgc cagggttttc ccagtcacga cgttgtaaaa cgacggccag
     2761 tcatgcataa tccgcacgca tctggaataa ggaagtgcca ttccgcctga cct
//
