LOCUS       sequence-316674-        2813 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cas9-mediated knockout of Limk1 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2813)
  AUTHORS   Aihara S, Fujimoto S, Sakaguchi R, Imai T
  TITLE     BMPR-2 gates activity-dependent stabilization of primary dendrites 
            during mitral cell remodeling.
  JOURNAL   Cell Rep. 2021 Jun 22;35(12):109276. doi: 
            10.1016/j.celrep.2021.109276.
  PUBMED    34161760
REFERENCE   2  (bases 1 to 2813)
  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: "Cell Rep.";
            date: "2021-06-22"; volume: "35(12):109276. doi"; pages: " 
            10.1016/j.celrep.2021.109276"
COMMENT     Sequence Label: Limk1 gRNA#1
FEATURES             Location/Qualifiers
     source          1..2813
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        73..321
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     73..93
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     244..263
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        342..417
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(429..453)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     misc_feature    659..715
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(728..744)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(728..744)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(741..763)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    752..768
                     /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(776..782,783..800,801..806))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    821..842
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(1130..1718)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1210..1229)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1889..2680,2681..2749))
                     /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     2512..2531
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(2750..2813,1..41))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
       61 ccacctgacg tcgagggcct atttcccatg attccttcat atttgcatat acgatacaag
      121 gctgttagag agataattag aattaatttg actgtaaaca caaagatatt agtacaaaat
      181 acgtgacgta gaaagtaata atttcttggg tagtttgcag ttttaaaatt atgttttaaa
      241 atggactatc atatgcttac cgtaacttga aagtatttcg atttcttggc tttatatatc
      301 ttgtggaaag gacgaaacac cgtggaaact tcattggcga tgttttagag ctagaaatag
      361 caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt
      421 ttttctagac ccagctttct tgtacaaagt tggcattaga tccgctgcat taatgaatcg
      481 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
      541 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
      601 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccaga
      661 attcgagctc ggtacccggg gatcctctag agtcgacctg caggcatgca agcttggcgt
      721 aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca
      781 tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat
      841 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
      901 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
      961 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     1021 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     1081 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     1141 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     1201 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     1261 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     1321 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     1381 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     1441 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     1501 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     1561 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     1621 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     1681 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     1741 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     1801 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     1861 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     1921 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     1981 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     2041 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     2101 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     2161 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     2221 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     2281 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     2341 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     2401 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     2461 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     2521 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     2581 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     2641 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     2701 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     2761 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac ata
//
