LOCUS       sequence-252923-        2917 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  sgRNA backbone for StCas9, combine with TaU3 promoter module 
            (pFH33).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2917)
  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 2917)
  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: pFH87
FEATURES             Location/Qualifiers
     source          1..2917
                     /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..731
                     /label=St1 gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus 
                     thermophilus CRISPR/Cas9 system (Esvelt et al., 2013)"
     primer_bind     complement(809..825)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     rep_origin      complement(997..1585)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1077..1096)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(1721..2536)
                     /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(1721..2536)
                     /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     2444..2463
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2528..2547
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     2657..2673
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2657..2673
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     terminator      2674..2720
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     2849..2865
                     /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 ttgtactctc aagatttaag taactgtaca acgaaactta cacagttact
      661 taaatcttgc agaagctaca aagataaggc ttcatgccga aatcaacacc ctgtcatttt
      721 atggcagggt gttttttttt cgcttgagac cgaggctagg tggaggctca gtgatgataa
      781 gtctgcgatg gtggatgcat gtgtcatggt catagctgtt tcctgtgtga aattgttatc
      841 cgctcagagg gcacaatcct attccgcgct atccgacaat ctccaagaca ttaggtggag
      901 ttcagttcgg cgtatggcat atgtcgctgg aaagaacatg tgagcaaaag gccagcaaaa
      961 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     1021 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     1081 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     1141 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     1201 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     1261 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     1321 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     1381 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac
     1441 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     1501 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     1561 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     1621 tctattcaac aaagccgccg tcccgtcaag tcagcgtaaa tgggtagggg gcttcaaatc
     1681 gtccgctctg ccagtgttac aaccaattaa caaattctga ttagaaaaac tcatcgagca
     1741 tcaaatgaaa ctgcaattta ttcatatcag gattatcaat accatatttt tgaaaaagcc
     1801 gtttctgtaa tgaaggagaa aactcaccga ggcagttcca taggatggca agatcctggt
     1861 atcggtctgc gattccgact cgtccaacat caatacaacc tattaatttc ccctcgtcaa
     1921 aaataaggtt atcaagtgag aaatcaccat gagtgacgac tgaatccggt gagaatggca
     1981 aaagcttatg catttctttc cagacttgtt caacaggcca gccattacgc tcgtcatcaa
     2041 aatcactcgc atcaaccaaa ccgttattca ttcgtgattg cgcctgagcg agacgaaata
     2101 cgcgatcgct gttaaaagga caattacaaa caggaatcga atgcaaccgg cgcaggaaca
     2161 ctgccagcgc atcaacaata ttttcacctg aatcaggata ttcttctaat acctggaatg
     2221 ctgttttccc ggggatcgca gtggtgagta accatgcatc atcaggagta cggataaaat
     2281 gcttgatggt cggaagaggc ataaattccg tcagccagtt tagtctgacc atctcatctg
     2341 taacatcatt ggcaacgcta cctttgccat gtttcagaaa caactctggc gcatcgggct
     2401 tcccatacaa tcgatagatt gtcgcacctg attgcccgac attatcgcga gcccatttat
     2461 acccatataa atcagcatcc atgttggaat ttaatcgcgg cctcgagcaa gacgtttccc
     2521 gttgaatatg gctcataaca ccccttgtat tactgtttat gtaagcagac agttttattg
     2581 ttcatgatga tatattttta tcttgtgcaa tgtaacatca gagattttga gacacaacgt
     2641 ggctttcccc cgccgctcta gaactagtgg atccaaataa aacgaaaggc tcagtcgaaa
     2701 gactgggcct ttcgttttat ctgttgtttg tcgcattata cgagacgtcc aggttgggat
     2761 acctgaaaca aaacccatcg tacggccaag gaagtctcca ataactgtga tccaccacaa
     2821 gcgccagggt tttcccagtc acgacgttgt aaaacgacgg ccagtcatgc ataatccgca
     2881 cgcatctgga ataaggaagt gccattccgc ctgacct
//
