LOCUS       sequence-267859-        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"
     terminator      103..189
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      281..308
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     316..339
                     /label=pENTR-F
                     /note="pENTR vectors, forward primer"
     protein_bind    358..457
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     509..525
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     509..525
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     regulatory      530..539
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             536..1225
                     /codon_start=1
                     /product="Anemonia majano cyan fluorescent protein"
                     /label=AmCyan1
                     /note="mammalian codon-optimized"
                     /translation="MALSNKFIGDDMKMTYHMDGCVNGHYFTVKGEGSGKPYEGTQTST
                     FKVTMANGGPLAFSFDILSTVFMYGNRCFTAYPTSMPDYFKQAFPDGMSYERTFTYEDG
                     GVATASWEISLKGNCFEHKSTFHGVNFPADGPVMAKKTTGWDPSFEKMTVCDGILKGDV
                     TAFLMLQGGGNYRCQFHTSYKTKKPVTMPPNHVVEHRIARTDLDKGGNSVQLTEHAVAH
                     ITSVVPF"
     primer_bind     complement(1255..1271)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1256..1272)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     protein_bind    complement(1282..1381)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1493..1512)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1504..2313
                     /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(1571..1590)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      2403..2991
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2892..2911
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgctagca tggatctcgg
       61 ggacgtctaa ctactaagcg agagtaggga actgccaggc atcaaataaa acgaaaggct
      121 cagtcggaag actgggcctt tcgttttatc tgttgtttgt cggtgaacgc tctcctgagt
      181 aggacaaatc cgccgggagc ggatttgaac gttgtgaagc aacggcccgg agggtggcgg
      241 gcaggacgcc cgccataaac tgccaggcat caaactaagc agaaggccat cctgacggat
      301 ggcctttttg cgtttctaca aactcttcct gttagttagt tacttaagct cgggccccaa
      361 ataatgattt tattttgact gatagtgacc tgttcgttgc aacaaattga taagcaatgc
      421 ttttttataa tgccaacttt gtacaaaaaa gcaggctggc gccggaacca attcagtcga
      481 ctggatccgg taccgaattc gcggccgctc tagaactagt ggatcccccg ccaccatggc
      541 cctgtccaac aagttcatcg gcgacgacat gaagatgacc taccacatgg acggctgcgt
      601 gaacggccac tacttcaccg tgaagggcga gggcagcggc aagccctacg agggcaccca
      661 gacctccacc ttcaaggtga ccatggccaa cggcggcccc ctggccttct ccttcgacat
      721 cctgtccacc gtgttcatgt acggcaaccg ctgcttcacc gcctacccca ccagcatgcc
      781 cgactacttc aagcaggcct tccccgacgg catgtcctac gagagaacct tcacctacga
      841 ggacggcggc gtggccaccg ccagctggga gatcagcctg aagggcaact gcttcgagca
      901 caagtccacc ttccacggcg tgaacttccc cgccgacggc cccgtgatgg ccaagaagac
      961 caccggctgg gacccctcct tcgagaagat gaccgtgtgc gacggcatct tgaagggcga
     1021 cgtgaccgcc ttcctgatgc tgcagggcgg cggcaactac agatgccagt tccacacctc
     1081 ctacaagacc aagaagcccg tgaccatgcc ccccaaccac gtggtggagc accgcatcgc
     1141 cagaaccgac ctggacaagg gcggcaacag cgtgcagctg accgagcacg ccgtggccca
     1201 catcacctcc gtggtgccct tctgagggct gcaggaattc gatatcaagc ttatcgatac
     1261 cgtcgacctc gagatatcta gacccagctt tcttgtacaa agttggcatt ataagaaagc
     1321 attgcttatc aatttgttgc aacgaacagg tcactatcag tcaaaataaa atcattattt
     1381 gccatccagc tgcagctctg gcccgtgtct caaaatctct gatgttacat tgcacaagat
     1441 aaaaatatat catcatgaac aataaaactg tctgcttaca taaacagtaa tacaaggggt
     1501 gttatgagcc atattcaacg ggaaacgtcg aggccgcgat taaattccaa catggatgct
     1561 gatttatatg ggtataaatg ggctcgcgat aatgtcgggc aatcaggtgc gacaatctat
     1621 cgcttgtatg ggaagcccga tgcgccagag ttgtttctga aacatggcaa aggtagcgtt
     1681 gccaatgatg ttacagatga gatggtcaga ctaaactggc tgacggaatt tatgcctctt
     1741 ccgaccatca agcattttat ccgtactcct gatgatgcat ggttactcac cactgcgatc
     1801 cccggaaaaa cagcattcca ggtattagaa gaatatcctg attcaggtga aaatattgtt
     1861 gatgcgctgg cagtgttcct gcgccggttg cattcgattc ctgtttgtaa ttgtcctttt
     1921 aacagcgatc gcgtatttcg tctcgctcag gcgcaatcac gaatgaataa cggtttggtt
     1981 gatgcgagtg attttgatga cgagcgtaat ggctggcctg ttgaacaagt ctggaaagaa
     2041 atgcataaac ttttgccatt ctcaccggat tcagtcgtca ctcatggtga tttctcactt
     2101 gataacctta tttttgacga ggggaaatta ataggttgta ttgatgttgg acgagtcgga
     2161 atcgcagacc gataccagga tcttgccatc ctatggaact gcctcggtga gttttctcct
     2221 tcattacaga aacggctttt tcaaaaatat ggtattgata atcctgatat gaataaattg
     2281 cagtttcatt tgatgctcga tgagtttttc taatcagaat tggttaattg gttgtaacat
     2341 tattcagatt gggccccgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     2401 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     2461 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     2521 ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac
     2581 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     2641 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     2701 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     2761 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     2821 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     2881 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     2941 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     3001 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gtt
//
