LOCUS       sequence-175363-        3684 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  sgRNA targeting GFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3684)
  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 3684)
  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-U6-sgGFP2-7SK-sgGFP3
FEATURES             Location/Qualifiers
     source          1..3684
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     89..108
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     342..359
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(518..545)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(637..723)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     772..794
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     786..803
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     787..803
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    819..918
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        944..1158
                     /label=H1 promoter
                     /note="human H1 RNA promoter"
     primer_bind     1079..1098
                     /label=H1
                     /note="Human H1 promoter, forward primer"
     misc_RNA        1185..1260
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        1306..1554
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1306..1326
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1477..1496
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        1575..1650
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_RNA        1946..2021
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(2059..2158)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2175..2194)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2176..2194)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2199..2215)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2199..2215)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2317..2336)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2328..3137
                     /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(2395..2414)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      join(3284..3684,1..188)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
       61 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
      121 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
      181 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
      241 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
      301 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
      361 aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat
      421 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaata
      481 cgcgtaccgc tagccaggaa gagtttgtag aaacgcaaaa aggccatccg tcaggatggc
      541 cttctgctta gtttgatgcc tggcagttta tggcgggcgt cctgcccgcc accctccggg
      601 ccgttgcttc acaacgttca aatccgctcc cggcggattt gtcctactca ggagagcgtt
      661 caccgacaaa caacagataa aacgaaaggc ccagtcttcc gactgagcct ttcgttttat
      721 ttgatgcctg gcagttccct actctcgcgt taacgctagc atggatgttt tcccagtcac
      781 gacgttgtaa aacgacggcc agtcttaagc tcgggcccca aataatgatt ttattttgac
      841 tgatagtgac ctgttcgttg caacaaattg atgagcaatg cttttttata atgccaactt
      901 tgtacaaaaa agcaggcttc atcgatcaat tgggatccaa ttcgaacgct gacgtcatca
      961 acccgctcca aggaatcgcg ggcccagtgt cactaggcgg gaacacccag cgcgcgtgcg
     1021 ccctggcagg aagatggctg tgagggacag gggagtggcg ccctgcaata tttgcatgtc
     1081 gctatgtgtt ctgggaaatc accataaacg tgaaatgtct ttggatttgg gaatcttata
     1141 agttctgtat gagaccacag atctgaagtt cgagggcgac acccgtttta gagctagaaa
     1201 tagcaagtta aaataaggct agtccgttat caacttgaaa aagtggcacc gagtcggtgc
     1261 ttttttagat ctggatccaa gcttccatgg aaggtcgggc aggaagaggg cctatttccc
     1321 atgattcctt catatttgca tatacgatac aaggctgtta gagagataat tagaattaat
     1381 ttgactgtaa acacaaagat attagtacaa aatacgtgac gtagaaagta ataatttctt
     1441 gggtagtttg cagttttaaa attatgtttt aaaatggact atcatatgct taccgtaact
     1501 tgaaagtatt tcgatttctt gggtttatat atcttgtgga aaggacgaaa caccgctgaa
     1561 gggcatcgac ttcagtttta gagctagaaa tagcaagtta aaataaggct agtccgttat
     1621 caacttgaaa aagtggcacc gagtcggtgc ttttttcata tgccatgggc ggccgctcta
     1681 gactgcagta tttagcatgc cccacccatc tgcaaggcat tctggatagt gtcaaaacag
     1741 ccggaaatca agtccgttta tctcaaactt tagcattttg ggaataaatg atatttgcta
     1801 tgctggttaa attagatttt agttaaattt cctgctgaag ctctagtacg ataagtaact
     1861 tgacctaagt gtaaagttga gatttccttc aggtttatat agcttgtgcg ccgcctgggt
     1921 acctccaact acaagacccg cgccggtttt agagctagaa atagcaagtt aaaataaggc
     1981 tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg cttttttggt acctctagac
     2041 tcgagactag taggcctgac ccagctttct tgtacaaagt tggcattata agaaagcatt
     2101 gcttatcaat ttgttgcaac gaacaggtca ctatcagtca aaataaaatc attatttgcc
     2161 atccagctga tatcccctat agtgagtcgt attacatggt catagctgtt tcctggcagc
     2221 tctggcccgt gtctcaaaat ctctgatgtt acattgcaca agataaaaat atatcatcat
     2281 gaacaataaa actgtctgct tacataaaca gtaatacaag gggtgttatg agccatattc
     2341 aacgggaaac gtcgaggccg cgattaaatt ccaacatgga tgctgattta tatgggtata
     2401 aatgggctcg cgataatgtc gggcaatcag gtgcgacaat ctatcgcttg tatgggaagc
     2461 ccgatgcgcc agagttgttt ctgaaacatg gcaaaggtag cgttgccaat gatgttacag
     2521 atgagatggt cagactaaac tggctgacgg aatttatgcc tcttccgacc atcaagcatt
     2581 ttatccgtac tcctgatgat gcatggttac tcaccactgc gatccccgga aaaacagcat
     2641 tccaggtatt agaagaatat cctgattcag gtgaaaatat tgttgatgcg ctggcagtgt
     2701 tcctgcgccg gttgcattcg attcctgttt gtaattgtcc ttttaacagc gatcgcgtat
     2761 ttcgtctcgc tcaggcgcaa tcacgaatga ataacggttt ggttgatgcg agtgattttg
     2821 atgacgagcg taatggctgg cctgttgaac aagtctggaa agaaatgcat aaacttttgc
     2881 cattctcacc ggattcagtc gtcactcatg gtgatttctc acttgataac cttatttttg
     2941 acgaggggaa attaataggt tgtattgatg ttggacgagt cggaatcgca gaccgatacc
     3001 aggatcttgc catcctatgg aactgcctcg gtgagttttc tccttcatta cagaaacggc
     3061 tttttcaaaa atatggtatt gataatcctg atatgaataa attgcagttt catttgatgc
     3121 tcgatgagtt tttctaatca gaattggtta attggttgta acactggcag agcattacgc
     3181 tgacttgacg ggacggcgca agctcatgac caaaatccct taacgtgagt tacgcgtcgt
     3241 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     3301 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     3361 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     3421 caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     3481 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     3541 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     3601 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     3661 acctacagcg tgagctatga gaaa
//
