LOCUS       sequence-175365-        3680 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway cloning.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3680)
  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 3680)
  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-sgHTT1-U6-sgGFP2-7SK-sgCas9
FEATURES             Location/Qualifiers
     source          1..3680
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(172..199)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(291..377)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     426..448
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     440..457
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     441..457
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    473..572
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        598..812
                     /label=H1 promoter
                     /note="human H1 RNA promoter"
     primer_bind     733..752
                     /label=H1
                     /note="Human H1 promoter, forward primer"
     misc_RNA        839..914
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        959..1207
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     959..979
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1130..1149
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        1228..1303
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_RNA        1597..1672
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(1709..1808)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1825..1844)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1826..1844)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1849..1865)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1849..1865)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1967..1986)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1978..2787
                     /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(2045..2064)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      2934..3522
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3423..3442
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     join(3676..3680,1..13)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg
       61 gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg
      121 caacgcaatt aatacgcgta ccgctagcca ggaagagttt gtagaaacgc aaaaaggcca
      181 tccgtcagga tggccttctg cttagtttga tgcctggcag tttatggcgg gcgtcctgcc
      241 cgccaccctc cgggccgttg cttcacaacg ttcaaatccg ctcccggcgg atttgtccta
      301 ctcaggagag cgttcaccga caaacaacag ataaaacgaa aggcccagtc ttccgactga
      361 gcctttcgtt ttatttgatg cctggcagtt ccctactctc gcgttaacgc tagcatggat
      421 gttttcccag tcacgacgtt gtaaaacgac ggccagtctt aagctcgggc cccaaataat
      481 gattttattt tgactgatag tgacctgttc gttgcaacaa attgatgagc aatgcttttt
      541 tataatgcca actttgtaca aaaaagcagg cttcatcgat caattgggat ccaattcgaa
      601 cgctgacgtc atcaacccgc tccaaggaat cgcgggccca gtgtcactag gcgggaacac
      661 ccagcgcgcg tgcgccctgg caggaagatg gctgtgaggg acaggggagt ggcgccctgc
      721 aatatttgca tgtcgctatg tgttctggga aatcaccata aacgtgaaat gtctttggat
      781 ttgggaatct tataagttct gtatgagacc acagatctga ccctggaaaa gctgatgagt
      841 tttagagcta gaaatagcaa gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg
      901 caccgagtcg gtgctttttt agatcggatc caagcttcca tggaaggtcg ggcaggaaga
      961 gggcctattt cccatgattc cttcatattt gcatatacga tacaaggctg ttagagagat
     1021 aattagaatt aatttgactg taaacacaaa gatattagta caaaatacgt gacgtagaaa
     1081 gtaataattt cttgggtagt ttgcagtttt aaaattatgt tttaaaatgg actatcatat
     1141 gcttaccgta acttgaaagt atttcgattt cttgggttta tatatcttgt ggaaaggacg
     1201 aaacaccgct gaagggcatc gacttcagtt ttagagctag aaatagcaag ttaaaataag
     1261 gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttc atatgccatg
     1321 ggcggccgct ctagactgca gtatttagca tgccccaccc atctgcaagg cattctggat
     1381 agtgtcaaaa cagccggaaa tcaagtccgt ttatctcaaa ctttagcatt ttgggaataa
     1441 atgatatttg ctatgctggt taaattagat tttagttaaa tttcctgctg aagctctagt
     1501 acgataagta acttgaccta agtgtaaagt tgagatttcc ttcaggttta tatagcttgt
     1561 gcgccgcctg ggtaccaatg gagtacttct tgtccagttt tagagctaga aatagcaagt
     1621 taaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gcttttttgg
     1681 tactctagac tcgagactag taggcctgac ccagctttct tgtacaaagt tggcattata
     1741 agaaagcatt gcttatcaat ttgttgcaac gaacaggtca ctatcagtca aaataaaatc
     1801 attatttgcc atccagctga tatcccctat agtgagtcgt attacatggt catagctgtt
     1861 tcctggcagc tctggcccgt gtctcaaaat ctctgatgtt acattgcaca agataaaaat
     1921 atatcatcat gaacaataaa actgtctgct tacataaaca gtaatacaag gggtgttatg
     1981 agccatattc aacgggaaac gtcgaggccg cgattaaatt ccaacatgga tgctgattta
     2041 tatgggtata aatgggctcg cgataatgtc gggcaatcag gtgcgacaat ctatcgcttg
     2101 tatgggaagc ccgatgcgcc agagttgttt ctgaaacatg gcaaaggtag cgttgccaat
     2161 gatgttacag atgagatggt cagactaaac tggctgacgg aatttatgcc tcttccgacc
     2221 atcaagcatt ttatccgtac tcctgatgat gcatggttac tcaccactgc gatccccgga
     2281 aaaacagcat tccaggtatt agaagaatat cctgattcag gtgaaaatat tgttgatgcg
     2341 ctggcagtgt tcctgcgccg gttgcattcg attcctgttt gtaattgtcc ttttaacagc
     2401 gatcgcgtat ttcgtctcgc tcaggcgcaa tcacgaatga ataacggttt ggttgatgcg
     2461 agtgattttg atgacgagcg taatggctgg cctgttgaac aagtctggaa agaaatgcat
     2521 aaacttttgc cattctcacc ggattcagtc gtcactcatg gtgatttctc acttgataac
     2581 cttatttttg acgaggggaa attaataggt tgtattgatg ttggacgagt cggaatcgca
     2641 gaccgatacc aggatcttgc catcctatgg aactgcctcg gtgagttttc tccttcatta
     2701 cagaaacggc tttttcaaaa atatggtatt gataatcctg atatgaataa attgcagttt
     2761 catttgatgc tcgatgagtt tttctaatca gaattggtta attggttgta acactggcag
     2821 agcattacgc tgacttgacg ggacggcgca agctcatgac caaaatccct taacgtgagt
     2881 tacgcgtcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     2941 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     3001 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     3061 gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac
     3121 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     3181 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     3241 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     3301 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     3361 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     3421 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     3481 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     3541 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
     3601 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
     3661 cgaacgaccg agcgcagcga
//
