LOCUS       sequence-156787-        2199 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  U6 promoter driven sgRNA expression plasmid. Annealed 
            oligonucleotides encoding crRNA sequence are ligated into BbsI 
            site..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2199)
  AUTHORS   Howden SE, McColl B, Glaser A, Vadolas J, Petrou S, Little MH, 
            Elefanty AG, Stanley EG
  TITLE     A Cas9 Variant for Efficient Generation of Indel-Free Knockin or 
            Gene-Corrected Human Pluripotent Stem Cells.
  JOURNAL   Stem Cell Reports. 2016 Sep 13;7(3):508-17. doi: 
            10.1016/j.stemcr.2016.07.001. Epub 2016 Aug 4.
  PUBMED    27499201
REFERENCE   2  (bases 1 to 2199)
  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"; doi: 
            "10.1016/j.stemcr.2016.07.001"; journalName: "Stem Cell Reports"; 
            date: "2016-09-13- 13"; volume: "7"; issue: "3"; pages: "508-17"
COMMENT     Sequence Label: pSMART-sgRNA (Sp)
FEATURES             Location/Qualifiers
     source          1..2199
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        42..290
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     42..62
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     213..232
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        316..391
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      484..515
                     /label=tonB terminator
                     /note="bidirectional E. coli tonB-P14 transcription 
                     terminator"
     promoter        516..618
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             619..1434
                     /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
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNGDRVFRLAQAQSRMNNGLVGA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(692..711)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      1455..1482
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     rep_origin      complement(1494..2081)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1574..1593)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(2103..2132)
                     /label=T3Te terminator
                     /note="phage T3 early transcription terminator"
ORIGIN
        1 ccaagctcgg atccgatatc gacgtcaagg tcgggcagga agagggccta tttcccatga
       61 ttccttcata tttgcatata cgatacaagg ctgttagaga gataattaga attaatttga
      121 ctgtaaacac aaagatatta gtacaaaata cgtgacgtag aaagtaataa tttcttgggt
      181 agtttgcagt tttaaaatta tgttttaaaa tggactatca tatgcttacc gtaacttgaa
      241 agtatttcga tttcttggct ttatatatct tgtggaaagg acgaaacacc gggtcttcat
      301 tattccagaa gacaagtttt agagctagaa atagcaagtt aaaataaggc tagtccgtta
      361 tcaacttgaa aaagtggcac cgagtcggtg cttttttttg cggccgcact tgaaattcgg
      421 acgaattctc tagatatcgc tcaatactga ccatttaaat catacctgac ctccatagca
      481 gaaagtcaaa agcctccgac cggaggcttt tgacttgatc ggcacgtaag aggttccaac
      541 tttcaccata atgaaataag atcactaccg ggcgtatttt ttgagttatc gagattttca
      601 ggagctaagg aagctaaaat gagccatatt caacgggaaa cgtcttgctc gaggccgcga
      661 ttaaattcca acatggatgc tgatttatat gggtataaat gggctcgcga taatgtcggg
      721 caatcaggtg cgacaatcta tcgattgtat gggaagcccg atgcgccaga gttgtttctg
      781 aaacatggca aaggtagcgt tgccaatgat gttacagatg agatggtcag gctaaactgg
      841 ctgacggaat ttatgcctct tccgaccatc aagcatttta tccgtactcc tgatgatgca
      901 tggttactca ccactgcgat cccagggaaa acagcattcc aggtattaga agaatatcct
      961 gattcaggtg aaaatattgt tgatgcgctg gcagtgttcc tgcgccggtt gcattcgatt
     1021 cctgtttgta attgtccttt taacggcgat cgcgtatttc gtctcgctca ggcgcaatca
     1081 cgaatgaata acggtttggt tggtgcgagt gattttgatg acgagcgtaa tggctggcct
     1141 gttgaacaag tctggaaaga aatgcataag cttttgccat tctcaccgga ttcagtcgtc
     1201 actcatggtg atttctcact tgataacctt atttttgacg aggggaaatt aataggttgt
     1261 attgatgttg gacgagtcgg aatcgcagac cgataccagg atcttgccat cctatggaac
     1321 tgcctcggtg agttttctcc ttcattacag aaacggcttt ttcaaaaata tggtattgat
     1381 aatcctgata tgaataaatt gcagtttcac ttgatgctcg atgagttttt ctgagggccc
     1441 aaatgtaatc acctggctca ccttcgggtg ggcctttctg cgttgctggc gtttttccat
     1501 aggctccgcc cccctgacga gcatcacaaa aatcgatgct caagtcagag gtggcgaaac
     1561 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     1621 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     1681 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     1741 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     1801 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     1861 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     1921 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt tacctcggaa
     1981 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     2041 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgattttct
     2101 accgaagaaa ggcccacccg tgaaggtgag ccagtgagtt gattgcagtc cagttacgct
     2161 ggagtctgag gctcgtcctg aatgatatca agcttgaat
//
