LOCUS       sequence-169990-        5695 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mouse Tubb3 target gRNA expression plasmid with CAGmCherry.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5695)
  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 5695)
  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..5695
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(42..629)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(122..141)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(801..1592,1593..1661))
                     /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     1424..1443
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1662..1766)
                     /gene="bla"
                     /label=AmpR promoter
     enhancer        1797..2176
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2178..2455
                     /label=chicken beta-actin promoter
     intron          2456..3468
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     3392..3414
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     3476..3495
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             3532..3558
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             3559..4266
                     /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(3701..3719)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3969..3988
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(4357..4376)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4422..4477
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4476..4495)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(4868..4884)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4868..4884)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4881..4903)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4892..4908
                     /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(4916..4922,4923..4940,4941..4946))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4961..4982
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        5109..5357
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     5109..5129
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     5280..5299
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        5378..5453
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(5465..5489)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(5566..5583)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
       61 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
      121 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
      181 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
      241 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
      301 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
      361 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
      421 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
      481 agacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
      541 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
      601 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
      661 acgctcagtg gaacgaaaac tcacgttaag ggattattgg tcatgagatt atcaaaaagg
      721 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
      781 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
      841 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
      901 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
      961 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     1021 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     1081 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     1141 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     1201 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     1261 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     1321 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     1381 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     1441 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     1501 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     1561 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     1621 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     1681 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     1741 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgggtcgaca
     1801 ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc atagcccata
     1861 tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga
     1921 cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt
     1981 ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt
     2041 gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca
     2101 ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt
     2161 catcgctatt accatggtcg aggtgagccc cacgttctgc ttcactctcc ccatctcccc
     2221 cccctcccca cccccaattt tgtatttatt tattttttaa ttattttgtg cagcgatggg
     2281 ggcggggggg gggggggggc gcgcgccagg cggggcgggg cggggcgagg ggcggggcgg
     2341 ggcgaggcgg agaggtgcgg cggcagccaa tcagagcggc gcgctccgaa agtttccttt
     2401 tatggcgagg cggcggcggc ggcggcccta taaaaagcga agcgcgcggc gggcgggagt
     2461 cgctgcgttg ccttcgcccc gtgccccgct ccgcgccgcc tcgcgccgcc cgccccggct
     2521 ctgactgacc gcgttactcc cacaggtgag cgggcgggac ggcccttctc ctccgggctg
     2581 taattagcgc ttggtttaat gacggctcgt ttcttttctg tggctgcgtg aaagccttaa
     2641 agggctccgg gagggccctt tgtgcggggg ggagcggctc ggggggtgcg tgcgtgtgtg
     2701 tgtgcgtggg gagcgccgcg tgcggcccgc gctgcccggc ggctgtgagc gctgcgggcg
     2761 cggcgcgggg ctttgtgcgc tccgcgtgtg cgcgagggga gcgcggccgg gggcggtgcc
     2821 ccgcggtgcg ggggggctgc gaggggaaca aaggctgcgt gcggggtgtg tgcgtggggg
     2881 ggtgagcagg gggtgtgggc gcggcggtcg ggctgtaacc cccccctgca cccccctccc
     2941 cgagttgctg agcacggccc ggcttcgggt gcggggctcc gtacggggcg tggcgcgggg
     3001 ctcgccgtgc cgggcggggg gtggcggcag gtgggggtgc cgggcggggc ggggccgcct
     3061 cgggccgggg agggctcggg ggaggggcgc ggcggccccc ggagcgccgg cggctgtcga
     3121 ggcgcggcga gccgcagcca ttgcctttta tggtaatcgt gcgagagggc gcagggactt
     3181 cctttgtccc aaatctgtgc ggagccgaaa tctgggaggc gccgccgcac cccctctagc
     3241 gggcgcgggg cgaagcggtg cggcgccggc aggaaggaaa tgggcgggga gggccttcgt
     3301 gcgtcgccgc gccgccgtcc ccttctccct ctccagcctc ggggctgtcc gcggggggac
     3361 ggctgccttc gggggggacg gggcagggcg gggttcggct tctggcgtgt gaccggcggc
     3421 tctagagcct ctgctaacca tgttcatgcc ttcttctttt tcctacagct cctgggcaac
     3481 gtgctggtta ttgtgctgtc tcatcatttt ggcaaagaat tgctcgagat gtatccgtat
     3541 gatgtgccgg attatgcggt gagcaagggc gaggaggata acatggccat catcaaggag
     3601 ttcatgcgct tcaaggtgca catggagggc tccgtgaacg gccacgagtt cgagatcgag
     3661 ggcgagggcg agggccgccc ctacgagggc acccagaccg ccaagctgaa ggtgaccaag
     3721 ggtggccccc tgcccttcgc ctgggacatc ctgtcccctc agttcatgta cggctccaag
     3781 gcctacgtga agcaccccgc cgacatcccc gactacttga agctgtcctt ccccgagggc
     3841 ttcaagtggg agcgcgtgat gaacttcgag gacggcggcg tggtgaccgt gacccaggac
     3901 tcctccctgc aggacggcga gttcatctac aaggtgaagc tgcgcggcac caacttcccc
     3961 tccgacggcc ccgtaatgca gaagaagacc atgggctggg aggcctcctc cgagcggatg
     4021 taccccgagg acggcgccct gaagggcgag atcaagcaga ggctgaagct gaaggacggc
     4081 ggccactacg acgctgaggt caagaccacc tacaaggcca agaagcccgt gcagctgccc
     4141 ggcgcctaca acgtcaacat caagttggac atcacctccc acaacgagga ctacaccatc
     4201 gtggaacagt acgaacgcgc cgagggccgc cactccaccg gcggcatgga cgagctgtac
     4261 aagtaagcgg ccgcaattca ctcctcaggt gcaggctgcc tatcagaagg tggtggctgg
     4321 tgtggccaat gccctggctc acaaatacca ctgagatctt tttccctctg ccaaaaatta
     4381 tggggacatc atgaagcccc ttgagcatct gacttctggc taataaagga aatttatttt
     4441 cattgcaata gtgtgttgga attttttgtg tctatcactc ggaaggacat atgggagggc
     4501 aaatcattta aaacatcaga atgagtattt ggtttagagt ttggcaacat atgcccatat
     4561 gctggctgcc atgaacaaag gttggctata aagaggtcat cagtatatga aacagccccc
     4621 tgctgtccat tccttattcc atagaaaagc cttgacttga ggttagattt tttttatatt
     4681 ttgttttgtg ttattttttt ctttaacatc cctaaaattt tccttacatg ttttactagc
     4741 cagatttttc ctcctctcct gactactccc agtcatagct gtccctcttc tcttatggag
     4801 atccctcgac ctgcaccgtc gaccagctgg tcgacggtgc accgtcgacc agcttggcgt
     4861 aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca
     4921 tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat
     4981 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagcggatct
     5041 gtacaaaaaa gcaggcttta aaggaaccaa ttcagtcgac tggatccggt accaaggtcg
     5101 ggcaggaaga gggcctattt cccatgattc cttcatattt gcatatacga tacaaggctg
     5161 ttagagagat aattagaatt aatttgactg taaacacaaa gatattagta caaaatacgt
     5221 gacgtagaaa gtaataattt cttgggtagt ttgcagtttt aaaattatgt tttaaaatgg
     5281 actatcatat gcttaccgta acttgaaagt atttcgattt cttggcttta tatatcttgt
     5341 ggaaaggacg aaacaccggc tgcgagcaac ttcacttgtt ttagagctag aaatagcaag
     5401 ttaaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttt
     5461 ctagacccag ctttcttgta caaagttggc attagatccg ctgcattaat gaatcggcca
     5521 acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc
     5581 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     5641 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccag
//
