LOCUS       sequence-171367-        3303 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  multiple sgRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3303)
  AUTHORS   Merienne N, Vachey G, de Longprez L, Meunier C, Zimmer V, Perriard 
            G, Canales M, Mathias A, Herrgott L, Beltraminelli T, Maulet A, 
            Dequesne T, Pythoud C, Rey M, Pellerin L, Brouillet E, Perrier AL, 
            du Pasquier R, Deglon N
  TITLE     The Self-Inactivating KamiCas9 System for the Editing of CNS Disease
            Genes.
  JOURNAL   Cell Rep. 2017 Sep 19;20(12):2980-2991. doi: 
            10.1016/j.celrep.2017.08.075.
  PUBMED    28930690
REFERENCE   2  (bases 1 to 3303)
  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.celrep.2017.08"; journalName: "Cell Rep"; date: 
            "2017-09-19- 19"; volume: "20"; issue: "12"; pages: "2980-2991"
COMMENT     Sequence Label: pENTR221-H1-sgGFP1-7SK-sgCas9
FEATURES             Location/Qualifiers
     source          1..3303
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     92..109
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(268..295)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(387..473)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     522..544
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     536..553
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     537..553
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    569..668
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        694..908
                     /label=H1 promoter
                     /note="human H1 RNA promoter"
     primer_bind     829..848
                     /label=H1
                     /note="Human H1 promoter, forward primer"
     misc_RNA        935..1010
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_RNA        1316..1391
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(1428..1527)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1544..1563)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1545..1563)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1568..1584)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1568..1584)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1686..1705)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1697..2506
                     /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="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(1764..1783)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      2653..3241
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3142..3161
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
       61 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
      121 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
      181 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaata cgcgtaccgc
      241 tagccaggaa gagtttgtag aaacgcaaaa aggccatccg tcaggatggc cttctgctta
      301 gtttgatgcc tggcagttta tggcgggcgt cctgcccgcc accctccggg ccgttgcttc
      361 acaacgttca aatccgctcc cggcggattt gtcctactca ggagagcgtt caccgacaaa
      421 caacagataa aacgaaaggc ccagtcttcc gactgagcct ttcgttttat ttgatgcctg
      481 gcagttccct actctcgcgt taacgctagc atggatgttt tcccagtcac gacgttgtaa
      541 aacgacggcc agtcttaagc tcgggcccca aataatgatt ttattttgac tgatagtgac
      601 ctgttcgttg caacaaattg atgagcaatg cttttttata atgccaactt tgtacaaaaa
      661 agcaggcttc atcgatcaat tgggatccaa ttcgaacgct gacgtcatca acccgctcca
      721 aggaatcgcg ggcccagtgt cactaggcgg gaacacccag cgcgcgtgcg ccctggcagg
      781 aagatggctg tgagggacag gggagtggcg ccctgcaata tttgcatgtc gctatgtgtt
      841 ctgggaaatc accataaacg tgaaatgtct ttggatttgg gaatcttata agttctgtat
      901 gagaccacag atctgaagtt cgagggcgac acccgtttta gagctagaaa tagcaagtta
      961 aaataaggct agtccgttat caacttgaaa aagtggcacc gagtcggtgc ttttttagat
     1021 ctggatccaa gcttccatgg gcggccgctc tagactgcag tatttagcat gccccaccca
     1081 tctgcaaggc attctggata gtgtcaaaac agccggaaat caagtccgtt tatctcaaac
     1141 tttagcattt tgggaataaa tgatatttgc tatgctggtt aaattagatt ttagttaaat
     1201 ttcctgctga agctctagta cgataagtaa cttgacctaa gtgtaaagtt gagatttcct
     1261 tcaggtttat atagcttgtg cgccgcctgg gtaccaatgg agtacttctt gtccagtttt
     1321 agagctagaa atagcaagtt aaaataaggc tagtccgtta tcaacttgaa aaagtggcac
     1381 cgagtcggtg cttttttggt actctagact cgagactagt aggcctgacc cagctttctt
     1441 gtacaaagtt ggcattataa gaaagcattg cttatcaatt tgttgcaacg aacaggtcac
     1501 tatcagtcaa aataaaatca ttatttgcca tccagctgat atcccctata gtgagtcgta
     1561 ttacatggtc atagctgttt cctggcagct ctggcccgtg tctcaaaatc tctgatgtta
     1621 cattgcacaa gataaaataa tatcatcatg aacaataaaa ctgtctgctt acataaacag
     1681 taatacaagg ggtgttatga gccatattca acgggaaacg tcgaggccgc gattaaattc
     1741 caacatggat gctgatttat atgggtataa atgggctcgc gataatgtcg ggcaatcagg
     1801 tgcgacaatc tatcgcttgt atgggaagcc cgatgcgcca gagttgtttc tgaaacatgg
     1861 caaaggtagc gttgccaatg atgttacaga tgagatggtc agactaaact ggctgacgga
     1921 atttatgcct cttccgacca tcaagcattt tatccgtact cctgatgatg catggttact
     1981 caccactgcg atccccggaa aaacagcatt ccaggtatta gaagaatatc ctgattcagg
     2041 tgaaaatatt gttgatgcgc tggcagtgtt cctgcgccgg ttgcattcga ttcctgtttg
     2101 taattgtcct tttaacagcg atcgcgtatt tcgtctcgct caggcgcaat cacgaatgaa
     2161 taacggtttg gttgatgcga gtgattttga tgacgagcgt aatggctggc ctgttgaaca
     2221 agtctggaaa gaaatgcata aacttttgcc attctcaccg gattcagtcg tcactcatgg
     2281 tgatttctca cttgataacc ttatttttga cgaggggaaa ttaataggtt gtattgatgt
     2341 tggacgagtc ggaatcgcag accgatacca ggatcttgcc atcctatgga actgcctcgg
     2401 tgagttttct ccttcattac agaaacggct ttttcaaaaa tatggtattg ataatcctga
     2461 tatgaataaa ttgcagtttc atttgatgct cgatgagttt ttctaatcag aattggttaa
     2521 ttggttgtaa cactggcaga gcattacgct gacttgacgg gacggcgcaa gctcatgacc
     2581 aaaatccctt aacgtgagtt acgcgtcgtt ccactgagcg tcagaccccg tagaaaagat
     2641 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     2701 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     2761 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt
     2821 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     2881 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     2941 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     3001 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     3061 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     3121 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     3181 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     3241 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     3301 gtt
//
