LOCUS       sequence-174887-        4833 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  mouse Tubb3 target gRNA expression plasmid with CAGmCherry.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 4833)
  AUTHORS   Suzuki K, Tsunekawa Y, Hernandez-Benitez R, Wu J, Zhu J, Kim EJ, 
            Hatanaka F, Yamamoto M, Araoka T, Li Z, Kurita M, Hishida T, Li M, 
            Aizawa E, Guo S, Chen S, Goebl A, Soligalla RD, Qu J, Jiang T, Fu X,
            Jafari M, Esteban CR, Berggren WT, Lajara J, Nunez-Delicado E, 
            Guillen P, Campistol JM, Matsuzaki F, Liu GH, Magistretti P, Zhang 
            K, Callaway EM, Zhang K, Belmonte JC
  TITLE     In vivo genome editing via CRISPR/Cas9 mediated homology-independent
            targeted integration.
  JOURNAL   Nature. 2016 Dec 1;540(7631):144-149. doi: 10.1038/nature20565. Epub
            2016 Nov 16.
  PUBMED    27851729
REFERENCE   2  (bases 1 to 4833)
  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"; doi: 
            "10.1038/nature20565"; journalName: "Nature"; date: "2016-12-1- 1"; 
            volume: "540"; issue: "7631"; pages: "144-149"
COMMENT     Sequence Label: Tubb3gRNA-mCherry
FEATURES             Location/Qualifiers
     source          1..4833
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     primer_bind     242..264
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     326..345
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             382..408
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             409..1116
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(551..569)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     819..838
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(1207..1226)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    1272..1327
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(1326..1345)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(1718..1734)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1718..1734)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1731..1753)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1742..1758
                     /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(1766..1772,1773..1790,1791..1796))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1811..1832
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        1959..2207
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1959..1979
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2130..2149
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2228..2303
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(2315..2339)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(2416..2433)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2587..3175)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2667..2686)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3346..4137,4138..4206))
                     /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     3969..3988
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4207..4311)
                     /gene="bla"
                     /label=AmpR promoter
     enhancer        4342..4721
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 tggtaatcgt gcgagagggc gcagggactt cctttgtccc aaatctgtgc ggagccgaaa
       61 tctgggaggc gccgccgcac cccctctagc gggcgcgggg cgaagcggtg cggcgccggc
      121 aggaaggaaa tgggcgggga gggccttcgt gcgtcgccgc gccgccgtcc ccttctccct
      181 ctccagcctc ggggctgtcc gcggggggac ggctgccttc gggggggacg gggcagggcg
      241 gggttcggct tctggcgtgt gaccggcggc tctagagcct ctgctaacca tgttcatgcc
      301 ttcttctttt tcctacagct cctgggcaac gtgctggtta ttgtgctgtc tcatcatttt
      361 ggcaaagaat tgctcgagat gtatccgtat gatgtgccgg attatgcggt gagcaagggc
      421 gaggaggata acatggccat catcaaggag ttcatgcgct tcaaggtgca catggagggc
      481 tccgtgaacg gccacgagtt cgagatcgag ggcgagggcg agggccgccc ctacgagggc
      541 acccagaccg ccaagctgaa ggtgaccaag ggtggccccc tgcccttcgc ctgggacatc
      601 ctgtcccctc agttcatgta cggctccaag gcctacgtga agcaccccgc cgacatcccc
      661 gactacttga agctgtcctt ccccgagggc ttcaagtggg agcgcgtgat gaacttcgag
      721 gacggcggcg tggtgaccgt gacccaggac tcctccctgc aggacggcga gttcatctac
      781 aaggtgaagc tgcgcggcac caacttcccc tccgacggcc ccgtaatgca gaagaagacc
      841 atgggctggg aggcctcctc cgagcggatg taccccgagg acggcgccct gaagggcgag
      901 atcaagcaga ggctgaagct gaaggacggc ggccactacg acgctgaggt caagaccacc
      961 tacaaggcca agaagcccgt gcagctgccc ggcgcctaca acgtcaacat caagttggac
     1021 atcacctccc acaacgagga ctacaccatc gtggaacagt acgaacgcgc cgagggccgc
     1081 cactccaccg gcggcatgga cgagctgtac aagtaagcgg ccgcaattca ctcctcaggt
     1141 gcaggctgcc tatcagaagg tggtggctgg tgtggccaat gccctggctc acaaatacca
     1201 ctgagatctt tttccctctg ccaaaaatta tggggacatc atgaagcccc ttgagcatct
     1261 gacttctggc taataaagga aatttatttt cattgcaata gtgtgttgga attttttgtg
     1321 tctctcactc ggaaggacat atgggagggc aaatcattta aaacatcaga atgagtattt
     1381 ggtttagagt ttggcaacat atgcccatat gctggctgcc atgaacaaag gttggctata
     1441 aagaggtcat cagtatatga aacagccccc tgctgtccat tccttattcc atagaaaagc
     1501 cttgacttga ggttagattt tttttatatt ttgttttgtg ttattttttt ctttaacatc
     1561 cctaaaattt tccttacatg ttttactagc cagatttttc ctcctctcct gactactccc
     1621 agtcatagct gtccctcttc tcttatggag atccctcgac ctgcaccgtc gaccagctgg
     1681 tcgacggtgc accgtcgacc agcttggcgt aatcatggtc atagctgttt cctgtgtgaa
     1741 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
     1801 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     1861 agtcgggaaa cctgtcgtgc cagcggatct gtacaaaaaa gcaggcttta aaggaaccaa
     1921 ttcagtcgac tggatccggt accaaggtcg ggcaggaaga gggcctattt cccatgattc
     1981 cttcatattt gcatatacga tacaaggctg ttagagagat aattagaatt aatttgactg
     2041 taaacacaaa gatattagta caaaatacgt gacgtagaaa gtaataattt cttgggtagt
     2101 ttgcagtttt aaaattatgt tttaaaatgg actatcatat gcttaccgta acttgaaagt
     2161 atttcgattt cttggcttta tatatcttgt ggaaaggacg aaacaccggc tgcgagcaac
     2221 ttcacttgtt ttagagctag aaatagcaag ttaaaataag gctagtccgt tatcaacttg
     2281 aaaaagtggc accgagtcgg tgcttttttt ctagacccag ctttcttgta caaagttggc
     2341 attagatccg ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     2401 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
     2461 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
     2521 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     2581 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     2641 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     2701 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     2761 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     2821 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     2881 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     2941 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     3001 gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc
     3061 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     3121 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     3181 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     3241 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
     3301 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     3361 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     3421 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     3481 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     3541 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     3601 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     3661 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     3721 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     3781 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     3841 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     3901 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     3961 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     4021 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     4081 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     4141 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     4201 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
     4261 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
     4321 ccgaaaagtg ccacctgggt cgacattgat tattgactag ttattaatag taatcaatta
     4381 cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg
     4441 gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc
     4501 ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa
     4561 ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca
     4621 atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta
     4681 cttggcagta catctacgta ttagtcatcg ctattaccat ggtcgaggtg agccccacgt
     4741 tctgcttcac tctccccatc tcccccccct ccccaccccc aattttgtat ttatttattt
     4801 tttaattatt ttgtgcagcg atgggggcgg ggg
//
