LOCUS pFH74 2543 bp DNA circular 30-MAY-2019 DEFINITION Subcloning of sgRNA protospacer/ tRNA-gRNA system with classic gRNA backbone/ Position 1. ACCESSION urn.local...28-aj37n96 KEYWORDS . SOURCE ORGANISM . FEATURES Location/Qualifiers misc_feature 66..71 /Recognition_pattern="GGTCTC(1/5)" /annotation_group="GGTCTC" /label="BsaI" /note="Geneious type: restriction site" overhang 73..76 /created_by="hahnf" /modified_by="hahnf" /label="TGGC overhang to pFH38" tRNA 77..153 /created_by="hahnf" /Color="#ff7400" /modified_by="hahnf" /label="tRNA Glycine" overhang 150..153 /created_by="koroleva" /label="TGCA" misc_feature complement(156..161) /Recognition_pattern="GAAGAC(2/6)" /annotation_group="GAAGAC" /label="BpiI" /note="Geneious type: restriction site" CDS 162..739 /created_by="koroleva" /label="LacZ" misc_feature 740..745 /Recognition_pattern="GAAGAC(2/6)" /annotation_group="GAAGAC" /label="BpiI" /note="Geneious type: restriction site" gRNA_Scaffold 748..777 /created_by="hahnf" /label="classic sgRNA backbone (part)" overhang 748..751 /created_by="koroleva" /label="GTTT" overhang 774..777 /created_by="koroleva" /label="AAAT" misc_feature complement(779..784) /Recognition_pattern="GGTCTC(1/5)" /annotation_group="GGTCTC" /label="BsaI" /note="Geneious type: restriction site" rep_origin complement(840..1454) /vntifkey=21 /Transferred_From="pAK-EL-01, pAK-EL-02, pAK-EL-03, pAK-EL-04, pAK-EL-05" /Transferred_Similarity="100.00%" /label="rep(pMB1)" CDS complement(1621..2430) /vntifkey=21 /Transferred_From="pAK-EL-01, pAK-EL-02, pAK-EL-03, pAK-EL-04, pAK-EL-05" /Transferred_Similarity="100.00%" /label="KanR" primer_bind 2501..2524 /Mismatches=0 /%GC="33.3" /Hairpin_Tm="None" /Self_Dimer_Tm="None" /Tm="56.0" /annotation_group="208495335923246c63475e-1bf1-45e4-a9ec-0 854b6af15c0" /Sequence="GCAATGTAACATCAGAGATTTTGA" /created_by="primer3" /label="FH32" ORIGIN 1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gacgtcattg 61 tcgatggtct catggcaaca aagcaccagt ggtctagtgg tagaatagta ccctgccacg 121 gtacagaccc gggttcgatt cccggctggt gcattgtctt cgcagctggc acgacaggtt 181 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta 241 ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg 301 ataacaattt cacacaggaa acagctatga ccatgattac gccaagcttg catgcctgca 361 ggtcgactct agaggatccc cgggtaccga gctcgaattc actggccgtc gttttacaac 421 gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt 481 tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca 541 gcctgaatgg cgaatggcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt 601 cacaccgcat atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagc 661 cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg 721 cttacagaca agctgtgacg aagacaagtt ttagagctag aaatagcaag ttaaaattga 781 gaccatcatt gtcacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc 841 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc 901 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga 961 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt 1021 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg 1081 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc 1141 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg 1201 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc 1261 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg 1321 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc 1381 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct 1441 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt 1501 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa 1561 aaatgaagtt ttaaatcaag cccaatctga ataatgttac aaccaattaa ccaattctga 1621 ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag gattatcaat 1681 accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga ggcagttcca 1741 taggatggca agatcctggt atcggtctgc gattccgact cgtccaacat caatacaacc 1801 tattaatttc ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat gagtgacgac 1861 tgaatccggt gagaatggca aaagtttatg catttctttc cagacttgtt caacaggcca 1921 gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca ttcgtgattg 1981 cgcctgagcg agacgaaata cgcgatcgct gttaaaagga caattacaaa caggaatcga 2041 atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata ttttcacctg aatcaggata 2101 ttcttctaat acctggaatg ctgtttttcc ggggatcgca gtggtgagta accatgcatc 2161 atcaggagta cggataaaat gcttgatggt cggaagaggc ataaattccg tcagccagtt 2221 tagtctgacc atctcatctg taacatcatt ggcaacgcta cctttgccat gtttcagaaa 2281 caactctggc gcatcgggct tcccatacaa gcgatagatt gtcgcacctg attgcccgac 2341 attatcgcga gcccatttat acccatataa atcagcatcc atgttggaat ttaatcgcgg 2401 cctcgacgtt tcccgttgaa tatggctcat aacacccctt gtattactgt ttatgtaagc 2461 agacagtttt attgttcatg atgatatatt tttatcttgt gcaatgtaac atcagagatt 2521 ttgagacacg ggccagagct gca //