LOCUS       sequence-413251-        3074 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  gRNA from a U6 promoter targeting the mouse ActB near the 3' end of 
            the ORF. Used for C-terminal knockin by DSB repair. [Lab plasmid ID:
            TU260].
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3074)
  AUTHORS   Richardson RR, Steyert M, Khim SN, Crutcher GW, Brandenburg C, 
            Robertson CD, Romanowski AJ, Inen J, Altas B, Poulopoulos A
  TITLE     Enhancing Precision and Efficiency of Cas9-Mediated Knockin Through 
            Combinatorial Fusions of DNA Repair Proteins.
  JOURNAL   CRISPR J. 2023 Oct;6(5):447-461. doi: 10.1089/crispr.2023.0036. Epub
            2023 Sep 15.
  PUBMED    37713292
REFERENCE   2  (bases 1 to 3074)
  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: 
            "10.1089/crispr.2023.0036"; journalName: "CRISPR J"; date: 
            "2023-10"; volume: "6"; issue: "5"; pages: "447-461"
COMMENT     Sequence Label: pS23.U6<ActB.mus_C-term-KI_gRNA
FEATURES             Location/Qualifiers
     source          1..3074
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(247..835)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(327..346)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1006..1797,1798..1866))
                     /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     1629..1648
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1867..1971)
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2657..2905
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2657..2677
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2828..2847
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
ORIGIN
        1 ggtctaatac ccaatctctc gtcttatcca gatgttttat acgccttaat taatagggat
       61 aacagggtaa tatcttgccc tgcacgaata ccagcgaccc cttgcccaaa tacttgccgt
      121 gggcctcggc ctgagagcca aaacacttga tgcggaagaa gtcggtgcgc tcctgcttgt
      181 cgccggcatc gttgcgccac atctaggatc tgccaggaac cgtaaaaagg ccgcgttgct
      241 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
      301 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
      361 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
      421 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
      481 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
      541 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
      601 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
      661 gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc
      721 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
      781 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
      841 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
      901 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
      961 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     1021 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     1081 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     1141 accgcgagaa ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     1201 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     1261 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     1321 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     1381 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     1441 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     1501 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     1561 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     1621 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     1681 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     1741 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     1801 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     1861 actcatactc ttcctttttc aatattattg aagcatttat caaggttatt gtctcatgag
     1921 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacgaattg
     1981 gccagcgctg cattaccctg ttatccctaa ccggtgcgtt tttatgcttg tagtattgta
     2041 taatgttttt aagatccgga gttctgcaat tcatgtgatc tgcgcctggg agctcacggg
     2101 gacagccccc ccccaaagcc cccagggatg taattacgtc cctcccccgc tagggggcag
     2161 cagcgagccg cccggggctc cgctccggtc cggcgctccc cccgcatccc cgagccggca
     2221 gcgtgcgggg acagcccggg cacggggaag gtggcacggg atcgctttcc tctgaacgct
     2281 tctcgctgct ctttgagcct gcagacacct ggggggatac ggggaaaaag ctaggctgaa
     2341 agaggatctt agaatgacac gcgcctggga gctcacgggg acagcccccc cccaaagccc
     2401 ccagggatgt aattacgtcc ctcccccgct agggggcagc agcgagccgc ccggggctcc
     2461 gctccggtcc ggcgctcccc ccgcatcccc gagccggcag cgtgcgggga cagcccgggc
     2521 acggggaagg tggcacggga tcgctttcct ctgaacgctt ctcgctgctc tttgagcctg
     2581 cagacacctg gggggatacg gggaaaaagc tggatcataa ctcgacgtgc atactgctgg
     2641 ccttttgctc acatgtgagg gcctatttcc catgattcct tcatatttgc atatacgata
     2701 caaggctgtt agagagataa ttggaattaa tttgactgta aacacaaaga tattagtaca
     2761 aaatacgtga cgtagaaagt aataatttct tgggtagttt gcagttttaa aattatgttt
     2821 taaaatggac tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct tggctttata
     2881 tatcttgtgg aaaggacgaa acaccagtcc gcctagaagc acttggtttc agagctatgc
     2941 tggaaacagc atagcaagtt gaaataaggc tagtccgtta tcaacttgaa aaagtggcac
     3001 cgagtcggtg cttttttgtt ttagagctag aaatagcaag ttaaaataag gctagtccgt
     3061 ttttagcgcg cgct
//
