LOCUS       sequence-341119-        6374 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 6374)
  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 6374)
  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..6374
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     intron          140..1156
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1080..1102
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1164..1183
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     primer_bind     complement(2889..2908)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    2954..3009
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3008..3027)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(3370..3386)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3370..3386)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3383..3405)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3394..3410
                     /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(3418..3424,3425..3442,3443..3448))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3463..3484
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        3543..3738
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      3589..3724
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    3744..3878
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3781..3800)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3835..3854
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3946..3963)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4117..4705)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4197..4216)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4876..5667,5668..5736))
                     /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     5499..5518
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5737..5841)
                     /gene="bla"
                     /label=AmpR promoter
     enhancer        5872..6251
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 ggggcggggc gaggggcggg gcggggcgag gcggagaggt gcggcggcag ccaatcagag
       61 cggcgcgctc cgaaagtttc cttttatggc gaggcggcgg cggcggcggc cctataaaaa
      121 gcgaagcgcg cggcgggcgg gagtcgctgc gcgctgcctt cgccccgtgc cccgctccgc
      181 cgccgcctcg cgccgcccgc cccggctctg actgaccgcg ttactcccac aggtgagcgg
      241 gcgggacggc ccttctcctc cgggctgtaa ttagcgcttg gtttaatgac ggcttgtttc
      301 ttttctgtgg ctgcgtgaaa gccttgaggg gctccgggag ggccctttgt gcggggggag
      361 cggctcgggg ggtgcgtgcg tgtgtgtgtg cgtggggagc gccgcgtgcg gctccgcgct
      421 gcccggcggc tgtgagcgct gcgggcgcgg cgcggggctt tgtgcgctcc gcagtgtgcg
      481 cgaggggagc gcggccgggg gcggtgcccc gcggtgcggg gggggctgcg aggggaacaa
      541 aggctgcgtg cggggtgtgt gcgtgggggg gtgagcaggg ggtgtgggcg cgtcggtcgg
      601 gctgcaaccc cccctgcacc cccctccccg agttgctgag cacggcccgg cttcgggtgc
      661 ggggctccgt acggggcgtg gcgcggggct cgccgtgccg ggcggggggt ggcggcaggt
      721 gggggtgccg ggcggggcgg ggccgcctcg ggccggggag ggctcggggg aggggcgcgg
      781 cggcccccgg agcgccggcg gctgtcgagg cgcggcgagc cgcagccatt gccttttatg
      841 gtaatcgtgc gagagggcgc agggacttcc tttgtcccaa atctgtgcgg agccgaaatc
      901 tgggaggcgc cgccgcaccc cctctagcgg gcgcggggcg aagcggtgcg gcgccggcag
      961 gaaggaaatg ggcggggagg gccttcgtgc gtcgccgcgc cgccgtcccc ttctccctct
     1021 ccagcctcgg ggctgtccgc ggggggacgg ctgccttcgg gggggacggg gcagggcggg
     1081 gttcggcttc tggcgtgtga ccggcggctc tagagcctct gctaaccatg ttcatgcctt
     1141 cttctttttc ctacagctcc tgggcaacgt gctggttatt gtgctgtctc atcattttgg
     1201 caaagaattc gccaccatga cttcctcgct gcatcggccc tttcgggtgc cctggctgct
     1261 atgggccgtc ctgctggtca gcactacggc tgcttctcag aatcaagaac ggctgtgtgc
     1321 atttaaagat ccatatcaac aagatcttgg gataggtgag agtcgaatct ctcatgaaaa
     1381 tgggacaata ttatgttcca aagggagcac gtgttatggt ctgtgggaga aatcaaaagg
     1441 ggacatcaat cttgtgaaac aaggatgttg gtctcacatc ggtgatcccc aagagtgcca
     1501 ctatgaagag tgtgtagtaa ctaccacccc accctcaatt cagaatggaa cgtaccgctt
     1561 ttgctgctgt agtacagatt tatgtaatgt caactttact gagaactttc caccccctga
     1621 cacaacacca ctcagtccac ctcattcatt taatcgagat gaaacgataa tcattgcttt
     1681 ggcatcagtt tctgtgttag ctgttttgat agtcgcctta tgttttggat acagaatgtt
     1741 gacaggagac cggaaacagg gtcttcacag catgaacatg atggaggcgg cagcagcaga
     1801 gccctccctt gacctggata acctgaagct gctggagctg attggacggg gtcgatacgg
     1861 agcagtatat aaaggttcct tggatgagcg tccagttgct gtaaaagtat tttcttttgc
     1921 aaaccgtcag aattttataa atgaaaaaaa catttacaga gtgcctttga tggaacatga
     1981 caacattgct cgcttcatag ttggagacga gaggctcact gcagacggcc gcatggagta
     2041 tttgcttgtg atggagtatt atcccaatgg atctctgtgc aaatatctga gtctccacac
     2101 aagtgattgg gtaagctctt gccgtctggc tcattctgtg actagaggac tggcttatct
     2161 tcacacagaa ttaccacgag gagatcatta taaacccgca atctcccacc gagatttaaa
     2221 cagcaggaat gtcctggtaa agaatgacgg cgcgtgtgtt atcagtgact ttggtttatc
     2281 catgaggcta actggaaatc ggctggtgcg cccaggggaa gaagataatg cggctataag
     2341 tgaggttggc acaattcgct atatggcacc agaagtgcta gaaggagctg tgaacctgag
     2401 ggactgtgag tcagctctga agcaagtgga catgtatgcg cttggactca tctactggga
     2461 ggtgtttatg aggtgtacag acctcttccc aggtgaatct gtaccagatt accagatggc
     2521 ttttcagaca gaagttggaa accatcccac atttgaggat atgcaggttc ttgtgtccag
     2581 agagaagcag agacccaagt tcccagaagc ctggaaagaa aatagcctgg cagtgaggtc
     2641 actcaaggaa acaattgaag actgctggga ccaggatgca gaggctcggc tcactgcaca
     2701 gtgtgctgag gagaggatgg ctgaactcat gatgatatgg gagagaaaca agtctgtgag
     2761 cccaacggtc aacccaatgt caactgctat gcagaatgaa cgcgcggccg cactcctcag
     2821 gtgcaggctg cctatcagaa ggtggtggct ggtgtggcca atgccctggc tcacaaatac
     2881 cactgagatc tttttccctc tgccaaaaat tatggggaca tcatgaagcc ccttgagcat
     2941 ctgacttctg gctaataaag gaaatttatt ttcattgcaa tagtgtgttg gaattttttg
     3001 tgtctctcac tcggaaggac atatgggagg gcaaatcatt taaaacatca gaatgagtat
     3061 ttggtttaga gtttggcaac atatgcccat atgctggctg ccatgaacaa aggttggcta
     3121 taaagaggtc atcagtatat gaaacagccc cctgctgtcc attccttatt ccatagaaaa
     3181 gccttgactt gaggttagat tttttttata ttttgttttg tgttattttt ttctttaaca
     3241 tccctaaaat tttccttaca tgttttacta gccagatttt tcctcctctc ctgactactc
     3301 ccagtcatag ctgtccctct tctcttatgg agatccctcg acctgcagcc caagcttggc
     3361 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa
     3421 catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac
     3481 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagcggat
     3541 ccgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta
     3601 actccgccca gttccgccca ttctccgccc catggctgac taattttttt tatttatgca
     3661 gaggccgagg ccgcctcggc ctctgagcta ttccagaagt agtgaggagg cttttttgga
     3721 ggcctaggct tttgcaaaaa gctaacttgt ttattgcagc ttataatggt tacaaataaa
     3781 gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt
     3841 tgtccaaact catcaatgta tcttatcatg tctggatccg ctgcattaat gaatcggcca
     3901 acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc
     3961 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     4021 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     4081 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     4141 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     4201 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     4261 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     4321 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     4381 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     4441 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     4501 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac
     4561 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     4621 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     4681 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     4741 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     4801 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     4861 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     4921 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     4981 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga
     5041 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     5101 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     5161 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     5221 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     5281 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     5341 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     5401 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     5461 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     5521 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     5581 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     5641 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     5701 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     5761 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     5821 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgggt cgacattgat
     5881 tattgactag ttattaatag taatcaatta cggggtcatt agttcatagc ccatatatgg
     5941 agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc
     6001 gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt
     6061 gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc
     6121 atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg
     6181 cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg
     6241 ctattaccat ggtcgaggtg agccccacgt tctgcttcac tctccccatc tcccccccct
     6301 ccccaccccc aattttgtat ttatttattt tttaattatt ttgtgcagcg atgggggcgg
     6361 gggggggggg gggg
//
