LOCUS       sequence-275567-        3053 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For cloning sgRNA flanked by two ribozyme elements, to subsequently 
            cloned into PL-5LTR-GW-A vector..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3053)
  AUTHORS   Gee P, Lung MSY, Okuzaki Y, Sasakawa N, Iguchi T, Makita Y, Hozumi 
            H, Miura Y, Yang LF, Iwasaki M, Wang XH, Waller MA, Shirai N, Abe 
            YO, Fujita Y, Watanabe K, Kagita A, Iwabuchi KA, Yasuda M, Xu H, 
            Noda T, Komano J, Sakurai H, Inukai N, Hotta A
  TITLE     Extracellular nanovesicles for packaging of CRISPR-Cas9 protein and 
            sgRNA to induce therapeutic exon skipping.
  JOURNAL   Nat Commun. 2020 Mar 13;11(1):1334. doi: 10.1038/s41467-020-14957-y.
  PUBMED    32170079
REFERENCE   2  (bases 1 to 3053)
  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"; journalName: "Nat 
            Commun."; date: "2020-03-13"; volume: "11(1):1334. doi"; pages: " 
            10.1038/s41467-020-14957-y"
COMMENT     Sequence Label: pENTR-AmCyan
FEATURES             Location/Qualifiers
     source          1..3053
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      277..286
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(291..307)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(291..307)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     protein_bind    complement(359..458)
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(477..500)
                     /label=pENTR-F
                     /note="pENTR vectors, forward primer"
     terminator      complement(508..535)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(627..713)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     rep_origin      complement(878..1466)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(958..977)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(1556..2365)
                     /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     2279..2298
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2357..2376
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     protein_bind    2488..2587
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     2597..2613
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     2598..2614
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             complement(join(2644..3053,1..280))
                     /codon_start=1
                     /product="Anemonia majano cyan fluorescent protein"
                     /label=AmCyan1
                     /note="mammalian codon-optimized"
                     /translation="MALSNKFIGDDMKMTYHMDGCVNGHYFTVKGEGSGKPYEGTQTST
                     FKVTMANGGPLAFSFDILSTVFMYGNRCFTAYPTSMPDYFKQAFPDGMSYERTFTYEDG
                     GVATASWEISLKGNCFEHKSTFHGVNFPADGPVMAKKTTGWDPSFEKMTVCDGILKGDV
                     TAFLMLQGGGNYRCQFHTSYKTKKPVTMPPNHVVEHRIARTDLDKGGNSVQLTEHAVAH
                     ITSVVPF"
ORIGIN
        1 acatgccgtc ggggaaggcc tgcttgaagt agtcgggcat gctggtgggg taggcggtga
       61 agcagcggtt gccgtacatg aacacggtgg acaggatgtc gaaggagaag gccagggggc
      121 cgccgttggc catggtcacc ttgaaggtgg aggtctgggt gccctcgtag ggcttgccgc
      181 tgccctcgcc cttcacggtg aagtagtggc cgttcacgca gccgtccatg tggtaggtca
      241 tcttcatgtc gtcgccgatg aacttgttgg acagggccat ggtggcgggg gatccactag
      301 ttctagagcg gccgcgaatt cggtaccgga tccagtcgac tgaattggtt ccggcgccag
      361 cctgcttttt tgtacaaagt tggcattata aaaaagcatt gcttatcaat ttgttgcaac
      421 gaacaggtca ctatcagtca aaataaaatc attatttggg gcccgagctt aagtaactaa
      481 ctaacaggaa gagtttgtag aaacgcaaaa aggccatccg tcaggatggc cttctgctta
      541 gtttgatgcc tggcagttta tggcgggcgt cctgcccgcc accctccggg ccgttgcttc
      601 acaacgttca aatccgctcc cggcggattt gtcctactca ggagagcgtt caccgacaaa
      661 caacagataa aacgaaaggc ccagtcttcc gactgagcct ttcgttttat ttgatgcctg
      721 gcagttccct actctcgctt agtagttaga cgtccccgag atccatgcta gcggtaatac
      781 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
      841 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
      901 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
      961 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     1021 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     1081 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     1141 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     1201 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     1261 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
     1321 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     1381 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     1441 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     1501 ctcagtggaa cggggcccaa tctgaataat gttacaacca attaaccaat tctgattaga
     1561 aaaactcatc gagcatcaaa tgaaactgca atttattcat atcaggatta tcaataccat
     1621 atttttgaaa aagccgtttc tgtaatgaag gagaaaactc accgaggcag ttccatagga
     1681 tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc aacatcaata caacctatta
     1741 atttcccctc gtcaaaaata aggttatcaa gtgagaaatc accatgagtg acgactgaat
     1801 ccggtgagaa tggcaaaagt ttatgcattt ctttccagac ttgttcaaca ggccagccat
     1861 tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct
     1921 gagcgagacg aaatacgcga tcgctgttaa aaggacaatt acaaacagga atcgaatgca
     1981 accggcgcag gaacactgcc agcgcatcaa caatattttc acctgaatca ggatattctt
     2041 ctaatacctg gaatgctgtt tttccgggga tcgcagtggt gagtaaccat gcatcatcag
     2101 gagtacggat aaaatgcttg atggtcggaa gaggcataaa ttccgtcagc cagtttagtc
     2161 tgaccatctc atctgtaaca tcattggcaa cgctaccttt gccatgtttc agaaacaact
     2221 ctggcgcatc gggcttccca tacaagcgat agattgtcgc acctgattgc ccgacattat
     2281 cgcgagccca tttataccca tataaatcag catccatgtt ggaatttaat cgcggcctcg
     2341 acgtttcccg ttgaatatgg ctcataacac cccttgtatt actgtttatg taagcagaca
     2401 gttttattgt tcatgatgat atatttttat cttgtgcaat gtaacatcag agattttgag
     2461 acacgggcca gagctgcagc tggatggcaa ataatgattt tattttgact gatagtgacc
     2521 tgttcgttgc aacaaattga taagcaatgc tttcttataa tgccaacttt gtacaagaaa
     2581 gctgggtcta gatatctcga ggtcgacggt atcgataagc ttgatatcga attcctgcag
     2641 ccctcagaag ggcaccacgg aggtgatgtg ggccacggcg tgctcggtca gctgcacgct
     2701 gttgccgccc ttgtccaggt cggttctggc gatgcggtgc tccaccacgt ggttgggggg
     2761 catggtcacg ggcttcttgg tcttgtagga ggtgtggaac tggcatctgt agttgccgcc
     2821 gccctgcagc atcaggaagg cggtcacgtc gcccttcaag atgccgtcgc acacggtcat
     2881 cttctcgaag gaggggtccc agccggtggt cttcttggcc atcacggggc cgtcggcggg
     2941 gaagttcacg ccgtggaagg tggacttgtg ctcgaagcag ttgcccttca ggctgatctc
     3001 ccagctggcg gtggccacgc cgccgtcctc gtaggtgaag gttctctcgt agg
//
