LOCUS       pFH99                   2868 bp    DNA     circular UNA 03-JUN-2019
DEFINITION  Subcloning of protospacer into SpCas9 classic sgRNA backbone,
            overhang to pFH35 AtU6-26p.
ACCESSION   urn.local...1r-ajo6si0
VERSION     urn.local...1r-ajo6si0
KEYWORDS    .
SOURCE      
  ORGANISM  
            .
FEATURES             Location/Qualifiers
     misc_feature    2..7
                     /Recognition_pattern="GGTCTC(1/5)"
                     /annotation_group="GGTCTC"
                     /label="BsaI"
                     /note="Geneious type: restriction site"
     overhang        9..12
                     /created_by="hahnf"
                     /modified_by="hahnf"
                     /label="ATTG to AtU6-26p pFH35"
     misc_feature    complement(15..20)
                     /Recognition_pattern="GAAGAC(2/6)"
                     /annotation_group="GAAGAC"
                     /label="BpiI"
                     /note="Geneious type: restriction site"
     CDS             21..598
                     /created_by="hahnf"
                     /label="lacZ"
     misc_feature    599..604
                     /Recognition_pattern="GAAGAC(2/6)"
                     /annotation_group="GAAGAC"
                     /label="BpiI"
                     /note="Geneious type: restriction site"
     gRNA_Scaffold   607..682
                     /created_by="hahnf"
                     /modified_by="hahnf"
                     /label="SpCas9 classic sgRNA scaffold"
     overhang        607..610
                     /created_by="hahnf"
                     /modified_by="hahnf"
                     /label="GTTT"
     Stop_codon      683..691
                     /created_by="hahnf"
                     /label="STOP"
     overhang        692..695
                     /created_by="hahnf"
                     /modified_by="hahnf"
                     /label="CGCT overhang to Level1 backbone"
     misc_feature    complement(697..702)
                     /Recognition_pattern="GGTCTC(1/5)"
                     /annotation_group="GGTCTC"
                     /label="BsaI"
                     /note="Geneious type: restriction site"
     rep_origin      complement(933..1547)
                     /vntifkey=21
                     /X-Transferred_From="pAK-EL-01; pAK-EL-02; pAK-EL-03;
                     pAK-EL-04; pAK-EL-05"
                     /X-Transferred_Similarity="100.00%"
                     /Transferred_From="pAK-EL-01, pAK-EL-02, pAK-EL-03,
                     pAK-EL-04, pAK-EL-05, pAK007 in pUC57-Kan-mini, pAK008 in
                     pUC57-Kan-mini, pAK009 in pUC57-Kan-mini, pAK010 in
                     pUC57-Kan-mini, pAK011 in pUC57-Kan-mini, pAK012 in
                     pUC57-Kan-mini"
                     /Transferred_Similarity="100.00%"
                     /label="rep(pMB1)"
     CDS             complement(1672..2487)
                     /vntifkey=21
                     /X-Transferred_From="pAK-EL-01; pAK-EL-02; pAK-EL-03;
                     pAK-EL-04; pAK-EL-05"
                     /X-Transferred_Similarity="100.00%"
                     /Transferred_From="pAK-EL-01, pAK-EL-02, pAK-EL-03,
                     pAK-EL-04, pAK-EL-05, pAK007 in pUC57-Kan-mini, pAK008 in
                     pUC57-Kan-mini, pAK009 in pUC57-Kan-mini, pAK010 in
                     pUC57-Kan-mini, pAK011 in pUC57-Kan-mini, pAK012 in
                     pUC57-Kan-mini"
                     /Transferred_Similarity="98.90%"
                     /label="KanR"
     primer_bind     2558..2581
                     /Mismatches=0
                     /%GC="33.3"
                     /Hairpin_Tm="None"
                     /Self_Dimer_Tm="None"
                     /Tm="56.0"
                     /annotation_group="24555775085359fbfbe37b-b3de-415d-988b-2
                     576bc743911"
                     /Sequence="GCAATGTAACATCAGAGATTTTGA"
                     /created_by="primer3"
                     /label="FH32"
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
//
