LOCUS       Exported                3303 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  multiple sgRNA.
ACCESSION   .
VERSION     .
KEYWORDS    pENTR221-H1-sgGFP1-7SK-sgCas9
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3303)
  AUTHORS   Merienne, Nicolas, Gabriel Vachey, Lucie de Longprez, Cecile 
            Meunier, Virginie Zimmer, Guillaume Perriard, Mathieu Canales, 
            Amandine Mathias, Lucas Herrgott, Tim Beltraminelli, Axelle Maulet, 
            Thomas Dequesne, Catherine Pythoud, Maria Rey, Luc Pellerin, 
            Emmanuel Brouillet, Anselme L. Perrier, Renaud du Pasquier,, Nicole 
            Deglon
  TITLE     The self-inactivating KamiCas9 system for the editing of CNS disease
            genes
  JOURNAL   Cell Reports , Volume 20 , Issue 12 , 2980 - 2991
REFERENCE   2  (bases 1 to 3303)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..3303
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     terminator      50..136
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     185..207
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     199..216
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     200..216
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    232..331
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        357..571
                     /label=H1 promoter
                     /note="human H1 RNA promoter"
     primer_bind     492..511
                     /label=H1
                     /note="Human H1 promoter, forward primer"
     misc_RNA        598..673
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_RNA        979..1054
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(1091..1190)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1207..1226)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1208..1226)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1231..1247)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1231..1247)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1349..1368)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1360..2169
                     /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(1427..1446)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      2316..2904
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2805..2824
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3058..3075
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      3234..3261
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
ORIGIN
        1 gccaccctcc gggccgttgc ttcacaacgt tcaaatccgc tcccggcgga tttgtcctac
       61 tcaggagagc gttcaccgac aaacaacaga taaaacgaaa ggcccagtct tccgactgag
      121 cctttcgttt tatttgatgc ctggcagttc cctactctcg cgttaacgct agcatggatg
      181 ttttcccagt cacgacgttg taaaacgacg gccagtctta agctcgggcc ccaaataatg
      241 attttatttt gactgatagt gacctgttcg ttgcaacaaa ttgatgagca atgctttttt
      301 ataatgccaa ctttgtacaa aaaagcaggc ttcatcgatc aattgggatc caattcgaac
      361 gctgacgtca tcaacccgct ccaaggaatc gcgggcccag tgtcactagg cgggaacacc
      421 cagcgcgcgt gcgccctggc aggaagatgg ctgtgaggga caggggagtg gcgccctgca
      481 atatttgcat gtcgctatgt gttctgggaa atcaccataa acgtgaaatg tctttggatt
      541 tgggaatctt ataagttctg tatgagacca cagatctgaa gttcgagggc gacacccgtt
      601 ttagagctag aaatagcaag ttaaaataag gctagtccgt tatcaacttg aaaaagtggc
      661 accgagtcgg tgctttttta gatctggatc caagcttcca tgggcggccg ctctagactg
      721 cagtatttag catgccccac ccatctgcaa ggcattctgg atagtgtcaa aacagccgga
      781 aatcaagtcc gtttatctca aactttagca ttttgggaat aaatgatatt tgctatgctg
      841 gttaaattag attttagtta aatttcctgc tgaagctcta gtacgataag taacttgacc
      901 taagtgtaaa gttgagattt ccttcaggtt tatatagctt gtgcgccgcc tgggtaccaa
      961 tggagtactt cttgtccagt tttagagcta gaaatagcaa gttaaaataa ggctagtccg
     1021 ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt ggtactctag actcgagact
     1081 agtaggcctg acccagcttt cttgtacaaa gttggcatta taagaaagca ttgcttatca
     1141 atttgttgca acgaacaggt cactatcagt caaaataaaa tcattatttg ccatccagct
     1201 gatatcccct atagtgagtc gtattacatg gtcatagctg tttcctggca gctctggccc
     1261 gtgtctcaaa atctctgatg ttacattgca caagataaaa atatatcatc atgaacaata
     1321 aaactgtctg cttacataaa cagtaataca aggggtgtta tgagccatat tcaacgggaa
     1381 acgtcgaggc cgcgattaaa ttccaacatg gatgctgatt tatatgggta taaatgggct
     1441 cgcgataatg tcgggcaatc aggtgcgaca atctatcgct tgtatgggaa gcccgatgcg
     1501 ccagagttgt ttctgaaaca tggcaaaggt agcgttgcca atgatgttac agatgagatg
     1561 gtcagactaa actggctgac ggaatttatg cctcttccga ccatcaagca ttttatccgt
     1621 actcctgatg atgcatggtt actcaccact gcgatccccg gaaaaacagc attccaggta
     1681 ttagaagaat atcctgattc aggtgaaaat attgttgatg cgctggcagt gttcctgcgc
     1741 cggttgcatt cgattcctgt ttgtaattgt ccttttaaca gcgatcgcgt atttcgtctc
     1801 gctcaggcgc aatcacgaat gaataacggt ttggttgatg cgagtgattt tgatgacgag
     1861 cgtaatggct ggcctgttga acaagtctgg aaagaaatgc ataaactttt gccattctca
     1921 ccggattcag tcgtcactca tggtgatttc tcacttgata accttatttt tgacgagggg
     1981 aaattaatag gttgtattga tgttggacga gtcggaatcg cagaccgata ccaggatctt
     2041 gccatcctat ggaactgcct cggtgagttt tctccttcat tacagaaacg gctttttcaa
     2101 aaatatggta ttgataatcc tgatatgaat aaattgcagt ttcatttgat gctcgatgag
     2161 tttttctaat cagaattggt taattggttg taacactggc agagcattac gctgacttga
     2221 cgggacggcg caagctcatg accaaaatcc cttaacgtga gttacgcgtc gttccactga
     2281 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     2341 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     2401 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     2461 gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     2521 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     2581 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     2641 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     2701 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     2761 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     2821 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     2881 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     2941 ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac
     3001 cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc
     3061 gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt
     3121 tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag
     3181 cgcaacgcaa ttaatacgcg taccgctagc caggaagagt ttgtagaaac gcaaaaaggc
     3241 catccgtcag gatggccttc tgcttagttt gatgcctggc agtttatggc gggcgtcctg
     3301 ccc
//
