LOCUS       sequence-275572-        6385 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses FKBP12 fused Gag for packaging FRB-SpCas9 into NanoMEDIC 
            particle..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6385)
  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 6385)
  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: pHLS-EF1a-FKBP12-Gag(HIV)
FEATURES             Location/Qualifiers
     source          1..6385
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..1179
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          232..1170
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1127..1147
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    1202..1226
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      1282..1291
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1336..1656
                     /codon_start=1
                     /product="human FK506-binding protein FKBP12"
                     /label=FKBP
                     /translation="GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKP
                     FKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELL
                     KLE"
     protein_bind    complement(3209..3233)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(3398..3417)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3463..3518
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3517..3536)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(3934..3951)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4105..4693)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4185..4204)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4864..5655,5656..5724))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     5487..5506
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5725..5829)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      6006..6141
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     6258..6276
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 ggctccggtg cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg
       61 ggaggggtcg gcaattgaac cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt
      121 gatgtcgtgt actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca
      181 gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc
      241 gtgtgtggtt cccgcgggcc tggcctcttt acgggttatg gcccttgcgt gccttgaatt
      301 acttccacct ggctgcagta cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg
      361 gagagttcga ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg
      421 cctgggcgct ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct
      481 ttcgataagt ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg
      541 caagatagtc ttgtaaatgc gggccaagat ctgcacactg gtatttcggt ttttggggcc
      601 gcgggcggcg acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga
      661 gcgcggccac cgagaatcgg acgggggtag tctcaagctg gccggcctgc tctggtgcct
      721 ggcctcgcgc cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca
      781 gttgcgtgag cggaaagatg gccgcttccc ggccctgctg cagggagctc aaaatggagg
      841 acgcggcgct cgggagagcg ggcgggtgag tcacccacac aaaggaaaag ggcctttccg
      901 tcctcagccg tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat
      961 tagttctcga gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg
     1021 gagtttcccc acactgagtg ggtggagact gaagttaggc cagcttggca cttgatgtaa
     1081 ttctccttgg aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca
     1141 gtggttcaaa gtttttttct tccatttcag gtgtcgtgag gaattagcgg tggcggccat
     1201 cacaagtttg tacaaaaaag caggctggcg ccggaaccaa ttcagtcgac tggatccggt
     1261 accgaattcg cggcccgaat tgccaccatg ggagctatta aatcaaaaag gaaagacgat
     1321 ccaccggcat ctagaggagt gcaggtggaa accatctccc caggagacgg gcgcaccttc
     1381 cccaagcgcg gccagacctg cgtggtgcac tacaccggga tgcttgaaga tggaaagaaa
     1441 tttgattcct cccgggacag aaacaagccc tttaagttta tgctaggcaa gcaggaggtg
     1501 atccgaggct gggaagaagg ggttgcccag atgagtgtgg gtcagagagc caaactgact
     1561 atatctccag attatgccta tggtgccact gggcacccag gcatcatccc accacatgcc
     1621 actctcgtct tcgatgtgga gcttctaaaa ctggaactgg ccgccagggc cagcgtgctg
     1681 agcggcggcg agctggacag gtgggagaag atcaggctga ggcccggcgg caagaagaag
     1741 tataagctga agcacatcgt gtgggccagc agggagctgg agaggttcgc cgtgaacccc
     1801 ggcctgctgg agaccagcga gggctgcagg cagatcctgg gccagctgca gcccagcctg
     1861 cagaccggca gcgaggagct gaggagcctg tacaacaccg tggccaccct gtactgcgtg
     1921 caccagagga tcgagatcaa ggacaccaag gaggccctgg acaagatcga ggaggagcag
     1981 aacaagtcca agaagaaggc ccagcaggcc gccgccgaca ccggccacag cagccaggtg
     2041 agccagaact accccatcgt gcagaacatc cagggccaga tggtgcacca ggccatcagc
     2101 cccaggaccc tgaacgcctg ggtgaaggtg gtggaggaga aggccttcag ccccgaggtg
     2161 atccccatgt tcagcgccct gagcgaggga gccacccccc aggacctgaa caccatgctg
     2221 aacaccgtgg gcggccacca ggccgccatg cagatgctga aggagaccat caacgaggag
     2281 gccgccgagt gggacagggt gcaccccgtg cacgccggcc ccatcgcccc cggccagatg
     2341 agggagcccc gcggcagcga catcgccggc accaccagca ccctgcagga gcagatcggc
     2401 tggatgacca acaacccccc catccccgtg ggcgaaatct acaagaggtg gatcatcctg
     2461 ggcctgaaca agatcgtgag gatgtacagc cccaccagca tcctggatat caggcagggc
     2521 cccaaagagc ccttcaggga ctacgtggac aggttctaca agaccctgcg cgccgagcag
     2581 gccagccagg aggtgaagaa ctggatgacc gagaccctgc tggtgcagaa cgccaacccc
     2641 gactgcaaga ccatcctgaa ggccctggga cccgccgcca ccctggagga gatgatgacc
     2701 gcctgccagg gcgtgggcgg ccccggccac aaggccaggg tgctggccga ggccatgagc
     2761 caggtgacca acaccgccac catcatgatg cagaggggca acttcaggaa ccagaggaag
     2821 atggtgaagt gcttcaactg cggcaaggag ggccacaccg ccaggaactg ccgcgccccc
     2881 aggaagaagg gctgctggaa gtgcggcaag gagggccacc agatgaagga ctgcaccgag
     2941 aggcaggcta attttttagg gaagatctgg ccttcctaca agggaaggcc agggaatttt
     3001 cttcagagca gaccagagcc aacagcccca ccatttcttc agagcagacc agagccaaca
     3061 gccccaccag aagagagctt caggtctggg gtagagacaa caactccccc tcagaagcag
     3121 gagccgatag acaaggaact gtatccttta acttccctca gatcactctt tggcaacgac
     3181 ccctcgtcac aatgatcgag atatctagac ccagctttct tgtacaaagt ggtgatctag
     3241 ctaaaacgcg gcctcgaatc gaattcgatt cgaatgtaca gatctggatc ctaggcttaa
     3301 gtatacctcg agttaaattc actcctcagg tgcaggctgc ctatcagaag gtggtggctg
     3361 gtgtggccaa tgccctggct cacaaatacc actgagatct ttttccctct gccaaaaatt
     3421 atggggacat catgaagccc cttgagcatc tgacttctgg ctaataaagg aaatttattt
     3481 tcattgcaat agtgtgttgg aattttttgt gtctctcact cggaaggaca tatgggaggg
     3541 caaatcattt aaaacatcag aatgagtatt tggtttagag tttggcaaca tatgcccata
     3601 tgctggctgc catgaacaaa ggttggctat aaagaggtca tcagtatatg aaacagcccc
     3661 ctgctgtcca ttccttattc catagaaaag ccttgacttg aggttagatt ttttttatat
     3721 tttgttttgt gttatttttt tctttaacat ccctaaaatt ttccttacat gttttactag
     3781 ccagattttt cctcctctcc tgactactcc cagtcatagc tgtccctctt ctcttatgga
     3841 gatccctcga cttaattaaa ccggtcagct gcattaatga atcggccaac gcgcggggag
     3901 aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt
     3961 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
     4021 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     4081 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     4141 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     4201 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     4261 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     4321 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     4381 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     4441 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     4501 tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat
     4561 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     4621 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     4681 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
     4741 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
     4801 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
     4861 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     4921 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     4981 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat
     5041 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     5101 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     5161 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     5221 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
     5281 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     5341 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     5401 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     5461 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
     5521 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     5581 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     5641 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     5701 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca
     5761 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     5821 ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc gctagcgttt aaacccgggc
     5881 gatcgctcta gattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     5941 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gggactagct ttttgcaaaa
     6001 gcctaggcct ccaaaaaagc ctcctcacta cttctggaat agctcagagg ccgaggcggc
     6061 ctcggcctct gcataaataa aaaaaattag tcagccatgg ggcggagaat gggcggaact
     6121 gggcggagtt aggggcggga tgggcggagt taggggcggg actatggttg ctgactaatt
     6181 gagatgagct tgcatgccga cattgattat tgactagtcc ctaagaaacc attcttatca
     6241 tgacattaac ctataaaaat aggcgtatca cgaggcctag actagtgcgg ccgctgcaga
     6301 agctttgcaa agatggataa agttttaaac agagaggaat ctttgcagct aatggacctt
     6361 ctaggtcttg aaaggagtgg gaatt
//
