LOCUS       sequence-174889-        4853 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  gRNA cloning vector with CAGmCherry.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 4853)
  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 4853)
  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: pCAGmCherry-gRNA
FEATURES             Location/Qualifiers
     source          1..4853
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     primer_bind     320..342
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     404..423
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             460..486
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             487..1194
                     /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(629..647)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     897..916
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(1285..1304)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    1350..1405
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(1404..1423)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(1796..1812)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1796..1812)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1809..1831)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1820..1836
                     /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(1844..1850,1851..1868,1869..1874))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1889..1910
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     2037..2057
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2208..2227
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(2334..2358)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(2435..2452)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2606..3194)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2686..2705)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3365..4156,4157..4225))
                     /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     3988..4007
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4226..4330)
                     /gene="bla"
                     /label=AmpR promoter
     enhancer        4361..4740
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 ggctcggggg aggggcgcgg cggcccccgg agcgccggcg gctgtcgagg cgcggcgagc
       61 cgcagccatt gccttttatg gtaatcgtgc gagagggcgc agggacttcc tttgtcccaa
      121 atctgtgcgg agccgaaatc tgggaggcgc cgccgcaccc cctctagcgg gcgcggggcg
      181 aagcggtgcg gcgccggcag gaaggaaatg ggcggggagg gccttcgtgc gtcgccgcgc
      241 cgccgtcccc ttctccctct ccagcctcgg ggctgtccgc ggggggacgg ctgccttcgg
      301 gggggacggg gcagggcggg gttcggcttc tggcgtgtga ccggcggctc tagagcctct
      361 gctaaccatg ttcatgcctt cttctttttc ctacagctcc tgggcaacgt gctggttatt
      421 gtgctgtctc atcattttgg caaagaattg ctcgagatgt atccgtatga tgtgccggat
      481 tatgcggtga gcaagggcga ggaggataac atggccatca tcaaggagtt catgcgcttc
      541 aaggtgcaca tggagggctc cgtgaacggc cacgagttcg agatcgaggg cgagggcgag
      601 ggccgcccct acgagggcac ccagaccgcc aagctgaagg tgaccaaggg tggccccctg
      661 cccttcgcct gggacatcct gtcccctcag ttcatgtacg gctccaaggc ctacgtgaag
      721 caccccgccg acatccccga ctacttgaag ctgtccttcc ccgagggctt caagtgggag
      781 cgcgtgatga acttcgagga cggcggcgtg gtgaccgtga cccaggactc ctccctgcag
      841 gacggcgagt tcatctacaa ggtgaagctg cgcggcacca acttcccctc cgacggcccc
      901 gtaatgcaga agaagaccat gggctgggag gcctcctccg agcggatgta ccccgaggac
      961 ggcgccctga agggcgagat caagcagagg ctgaagctga aggacggcgg ccactacgac
     1021 gctgaggtca agaccaccta caaggccaag aagcccgtgc agctgcccgg cgcctacaac
     1081 gtcaacatca agttggacat cacctcccac aacgaggact acaccatcgt ggaacagtac
     1141 gaacgcgccg agggccgcca ctccaccggc ggcatggacg agctgtacaa gtaagcggcc
     1201 gcaattcact cctcaggtgc aggctgccta tcagaaggtg gtggctggtg tggccaatgc
     1261 cctggctcac aaataccact gagatctttt tccctctgcc aaaaattatg gggacatcat
     1321 gaagcccctt gagcatctga cttctggcta ataaaggaaa tttattttca ttgcaatagt
     1381 gtgttggaat tttttgtgtc tctcactcgg aaggacatat gggagggcaa atcatttaaa
     1441 acatcagaat gagtatttgg tttagagttt ggcaacatat gcccatatgc tggctgccat
     1501 gaacaaaggt tggctataaa gaggtcatca gtatatgaaa cagccccctg ctgtccattc
     1561 cttattccat agaaaagcct tgacttgagg ttagattttt tttatatttt gttttgtgtt
     1621 atttttttct ttaacatccc taaaattttc cttacatgtt ttactagcca gatttttcct
     1681 cctctcctga ctactcccag tcatagctgt ccctcttctc ttatggagat ccctcgacct
     1741 gcaccgtcga ccagctggtc gacggtgcac cgtcgaccag cttggcgtaa tcatggtcat
     1801 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa
     1861 gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc
     1921 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gcggatctgt acaaaaaagc
     1981 aggctttaaa ggaaccaatt cagtcgactg gatccggtac caaggtcggg caggaagagg
     2041 gcctatttcc catgattcct tcatatttgc atatacgata caaggctgtt agagagataa
     2101 ttagaattaa tttgactgta aacacaaaga tattagtaca aaatacgtga cgtagaaagt
     2161 aataatttct tgggtagttt gcagttttaa aattatgttt taaaatggac tatcatatgc
     2221 ttaccgtaac ttgaaagtat ttcgatttct tggctttata tatcttaagt taaaataagg
     2281 ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gctttttttc tagacccagc
     2341 tttcttgtac aaagttggca ttagatccgc tgcattaatg aatcggccaa cgcgcgggga
     2401 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     2461 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     2521 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     2581 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     2641 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     2701 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     2761 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     2821 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     2881 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     2941 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     3001 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
     3061 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     3121 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     3181 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     3241 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     3301 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     3361 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     3421 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     3481 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     3541 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     3601 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     3661 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     3721 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     3781 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     3841 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     3901 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     3961 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     4021 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     4081 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     4141 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     4201 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     4261 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     4321 gggttccgcg cacatttccc cgaaaagtgc cacctgggtc gacattgatt attgactagt
     4381 tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt
     4441 acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg
     4501 tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg
     4561 gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt
     4621 acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg
     4681 accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg
     4741 gtcgaggtga gccccacgtt ctgcttcact ctccccatct cccccccctc cccaccccca
     4801 attttgtatt tatttatttt ttaattattt tgtgcagcga tgggggcggg ggg
//
