LOCUS       sequence-266143-        5181 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Empty backbone for cloning sgRNA sequence to be used in nanoblades 
            system.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5181)
  AUTHORS   Mangeot PE, Risson V, Fusil F, Marnef A, Laurent E, Blin J, 
            Mournetas V, Massourides E, Sohier TJM, Corbin A, Aube F, Teixeira 
            M, Pinset C, Schaeffer L, Legube G, Cosset FL, Verhoeyen E, Ohlmann 
            T, Ricci EP
  TITLE     Genome editing in primary cells and in vivo using viral-derived 
            Nanoblades loaded with Cas9-sgRNA ribonucleoproteins.
  JOURNAL   Nat Commun. 2019 Jan 3;10(1):45. doi: 10.1038/s41467-018-07845-z.
  PUBMED    30604748
REFERENCE   2  (bases 1 to 5181)
  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: "2019-01-3"; volume: "10(1):45. doi"; pages: " 
            10.1038/s41467-018-07845-z"
COMMENT     Sequence Label: BLADE
FEATURES             Location/Qualifiers
     source          1..5181
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(30..458)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(140..161)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     352..371
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     585..607
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     599..616
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     600..616
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     623..642
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        623..641
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     674..690
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     674..690
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     promoter        706..954
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     706..726
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     877..896
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2836..2911
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(2993..3011)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3032..3048)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3032..3048)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3045..3067)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3056..3072
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(3080..3086,3087..3104,3105..3110))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3125..3146
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3263..3280)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3434..4022)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3514..3533)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4193..4984,4985..5053))
                     /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     4816..4835
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5054..5158)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
       61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      121 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
      181 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
      241 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
      301 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
      361 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
      421 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg
      481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
      541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
      601 taaaacgacg gccagtgaat tgtaatacga ctcactatag ggcgaattgg agctccaccg
      661 cggtggcggc cgctctagaa ctagtggatc ccagggggac ccagagaggg cctatttccc
      721 atgattcctt catatttgca tatacgatac aaggctgtta gagagataat tagaattaat
      781 ttgactgtaa acacaaagat attagtacaa aatacgtgac gtagaaagta ataatttctt
      841 gggtagtttg cagttttaaa attatgtttt aaaatggact atcatatgct taccgtaact
      901 tgaaagtatt tcgatttctt ggctttatat atcttgtgga aaggacgaaa caccggagac
      961 ggttgtaaat gagcacacaa aatacacatg ctaaaatatt atattctatg acctttataa
     1021 aatcaaccaa aatcttcttt ttaataactt tagtatcaat aattagaatt tttatgttcc
     1081 tttttgcaaa cttttaataa aaatgagcaa aataaaaaaa cgctagtttt agtaactcgc
     1141 gttgttttct tcacctttaa taatagctac tccaccactt gttcctaagc ggtcagctcc
     1201 tgcttcaatc attttttgag catcttcaaa tgttctaact ccaccagctg ctttaactaa
     1261 agcattgtct ttaacaactg acttcattag tttaacatct tcaaatgttg cacctgattt
     1321 tgaaaatcct gttgatgttt taacaaattc taatccagct tcaacagcta tttcacaagc
     1381 tttcatgatt tcttcttttg ttaataaaca attttccata atacatttaa caacatgtga
     1441 tccagctgct ttttttacag ctttcatgtc ttctaaaact aattcataat ttttgtcttt
     1501 taatgcacca atatttaata ccatatcaat ttctgttgca ccatctttaa ttgcttcaga
     1561 aacttcgaat gcttttgtag ctgttgtgca tgcacctaga ggaaaaccta caacatttgt
     1621 tattcctaca tttgtgcctt ttaataattc tttacaatag cttgttcaat atgaattaac
     1681 acaaactgtt gcaaaatcaa attcaattgc ttcatcacat aattgtttaa tttcagcttt
     1741 cgtagcatct tgttttaata atgtgtgatc tatatatttg tttagtttca ttttttctcc
     1801 tatatattca tttttaattt taattcttta ataatttcgt ctactttaac tttagcgtat
     1861 tgaacagatt caccaacacc tataaaataa atttttagtt taggttcagt tccacttggg
     1921 cgaacagcaa atcatgactt atcttctaaa taaaatttta gtaagtcttg tcctggcata
     1981 ttatacattc catcgatgta gtcttcaaca ttaacaactt taagtccagc aatttgagtt
     2041 aagggtgttg ctctcaatga tttcattaat ggttcaattt ttaatttctt ttcttctggt
     2101 ttaaaattca agtttaaagt gaaagtgtaa tatgcaccca tttctttaaa taaatcttct
     2161 aaatagtcta ctaatgtttt attttgtttt ttataaaatc aagcagcctc tgctattaat
     2221 atagaagctt gtattccatc tttatctcta gctgagtcat caattacata tccataactt
     2281 tcttcataag caaaaacaaa atttaatccg ttatcttctt ctttagcaat ttctctaccc
     2341 attcatttaa atccagttaa agtttttaca atattaactc catatttttc atgagcgatt
     2401 ctatcaccca aatcacttgt tacaaaactt gaatatagag ccggattttt tggaatgcta
     2461 tttaagcgtt ttagatttga taattttcaa tcaattaaaa ttggtcctgt ttgatttcca
     2521 tctaatctta caaaatgacc atcatgtttt attgccattc caaatctgtc agcatctggg
     2581 tcattcataa taataatatc tgcatcatgt ttaataccat attcaagcgg tatttttcat
     2641 gcaggatcaa attctggatt tggatttaca acatttttaa atgtttcatc ttcaaatgca
     2701 tgctcttcaa cctcaataac gttatatcct gattcacgta atatttttgg ggtaaattta
     2761 gttcctgttc cattaactgc gctaaaaata atttttaaat cttttttagc ttcttgctct
     2821 tttttgtacg tctctgtttt agagctagaa atagcaagtt aaaataaggc tagtccgtta
     2881 tcaacttgaa aaagtggcac cgagtcggtg cttttttgaa ttctagatct tgagacaaat
     2941 ggcagtattc atccacaatt ctcgaggggg ggcccggtac cagcttttgt tccctttagt
     3001 gagggttaat ttcgagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt
     3061 atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg
     3121 cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg
     3181 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     3241 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     3301 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     3361 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     3421 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     3481 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     3541 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     3601 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     3661 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     3721 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     3781 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     3841 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
     3901 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     3961 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     4021 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     4081 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     4141 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     4201 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     4261 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     4321 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     4381 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     4441 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     4501 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     4561 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     4621 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     4681 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     4741 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     4801 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     4861 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     4921 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     4981 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     5041 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     5101 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     5161 catttccccg aaaagtgcca c
//
