LOCUS       sequence-266137-        3321 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  sgRNA targeting GFP to be used in nanoblade system.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3321)
  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 3321)
  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-182
FEATURES             Location/Qualifiers
     source          1..3321
                     /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        976..1051
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(1133..1151)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1172..1188)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1172..1188)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1185..1207)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1196..1212
                     /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(1220..1226,1227..1244,1245..1250))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1265..1286
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1403..1420)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1574..2162)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1654..1673)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2333..3124,3125..3193))
                     /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     2956..2975
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3194..3298)
                     /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 caccgcgagg
      961 agctgttcac cgggggtttt agagctagaa atagcaagtt aaaataaggc tagtccgtta
     1021 tcaacttgaa aaagtggcac cgagtcggtg cttttttgaa ttctagatct tgagacaaat
     1081 ggcagtattc atccacaatt ctcgaggggg ggcccggtac cagcttttgt tccctttagt
     1141 gagggttaat ttcgagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt
     1201 atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg
     1261 cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg
     1321 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     1381 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     1441 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     1501 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     1561 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     1621 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     1681 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     1741 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     1801 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     1861 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     1921 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     1981 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
     2041 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     2101 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     2161 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     2221 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     2281 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     2341 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     2401 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     2461 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     2521 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     2581 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     2641 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     2701 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     2761 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     2821 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     2881 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     2941 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     3001 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     3061 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     3121 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     3181 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     3241 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     3301 catttccccg aaaagtgcca c
//
