LOCUS       sequence-394343-        2812 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cas9-mediated knockout of RhoA in mammalian cells #3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2812)
  AUTHORS   Fujimoto S, Leiwe MN, Aihara S, Sakaguchi R, Muroyama Y, Kobayakawa 
            R, Kobayakawa K, Saito T, Imai T
  TITLE     Activity-dependent local protection and lateral inhibition control 
            synaptic competition in developing mitral cells in mice.
  JOURNAL   Dev Cell. 2023 Jul 24;58(14):1221-1236.e7. doi: 
            10.1016/j.devcel.2023.05.004. Epub 2023 Jun 7.
  PUBMED    37290446
REFERENCE   2  (bases 1 to 2812)
  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: 
            "10.1016/j.devcel.2023.05.004"; journalName: "Dev Cell"; date: 
            "2023-07-24- 24"; volume: "58"; issue: "14"; pages: "1221-1236.e7"
COMMENT     Sequence Label: RhoA gRNA #3
FEATURES             Location/Qualifiers
     source          1..2812
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        72..320
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     72..92
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     243..262
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        341..416
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(428..452)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     misc_feature    658..714
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(727..743)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(727..743)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(740..762)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    751..767
                     /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(775..781,782..799,800..805))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    820..841
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(1129..1717)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1209..1228)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1888..2679,2680..2748))
                     /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     2511..2530
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(2749..2812,1..41))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
       61 ccacctgacg tgagggccta tttcccatga ttccttcata tttgcatata cgatacaagg
      121 ctgttagaga gataattaga attaatttga ctgtaaacac aaagatatta gtacaaaata
      181 cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta tgttttaaaa
      241 tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct ttatatatct
      301 tgtggaaagg acgaaacacc gcgtgtgctc gtcattccga gttttagagc tagaaatagc
      361 aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt
      421 tttctagacc cagctttctt gtacaaagtt ggcattagat ccgctgcatt aatgaatcgg
      481 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga
      541 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
      601 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagaa
      661 ttcgagctcg gtacccgggg atcctctaga gtcgacctgc aggcatgcaa gcttggcgta
      721 atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat
      781 acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt
      841 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
      901 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc
      961 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     1021 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     1081 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     1141 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     1201 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     1261 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     1321 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     1381 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     1441 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     1501 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     1561 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     1621 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     1681 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     1741 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     1801 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     1861 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     1921 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     1981 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     2041 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     2101 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     2161 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     2221 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     2281 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     2341 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     2401 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     2461 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     2521 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     2581 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     2641 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     2701 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     2761 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca ta
//
