LOCUS       sequence-394350-        5384 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses gRNA resistant RhoA under the CAG promoter in mammalian 
            cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5384)
  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 5384)
  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: pCAG-RhoA*
FEATURES             Location/Qualifiers
     source          1..5384
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        5..384
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        386..661
                     /label=chicken beta-actin promoter
     intron          662..1678
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1602..1624
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1686..1705
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      1733..1742
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(2406..2425)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    2471..2526
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(2525..2544)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(2887..2903)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2887..2903)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2900..2922)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2911..2927
                     /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(2935..2941,2942..2959,2960..2965))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2980..3001
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        3060..3255
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      3106..3241
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    3261..3395
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3298..3317)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3352..3371
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3463..3480)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3634..4222)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3714..3733)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4393..5184,5185..5253))
                     /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     5016..5035
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5254..5358)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ggtcgacatt gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat
       61 agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg
      121 cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata
      181 gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta
      241 catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc
      301 gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca gtacatctac
      361 gtattagtca tcgctattac catggtcgag gtgagcccca cgttctgctt cactctcccc
      421 atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca
      481 gcgatggggg cggggggggg gggggsgcgc gccaggcggg gcggggcggg gcgaggggcg
      541 gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt
      601 tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc
      661 gggagtcgct gcgcgctgcc ttcgccccgt gccccgctcc gccgccgcct cgcgccgccc
      721 gccccggctc tgactgaccg cgttactccc acaggtgagc gggcgggacg gcccttctcc
      781 tccgggctgt aattagcgct tggtttaatg acggcttgtt tcttttctgt ggctgcgtga
      841 aagccttgag gggctccggg agggcccttt gtgcgggggg agcggctcgg ggggtgcgtg
      901 cgtgtgtgtg tgcgtgggga gcgccgcgtg cggctccgcg ctgcccggcg gctgtgagcg
      961 ctgcgggcgc ggcgcggggc tttgtgcgct ccgcagtgtg cgcgagggga gcgcggccgg
     1021 gggcggtgcc ccgcggtgcg gggggggctg cgaggggaac aaaggctgcg tgcggggtgt
     1081 gtgcgtgggg gggtgagcag ggggtgtggg cgcgtcggtc gggctgcaac cccccctgca
     1141 cccccctccc cgagttgctg agcacggccc ggcttcgggt gcggggctcc gtacggggcg
     1201 tggcgcgggg ctcgccgtgc cgggcggggg gtggcggcag gtgggggtgc cgggcggggc
     1261 ggggccgcct cgggccgggg agggctcggg ggaggggcgc ggcggccccc ggagcgccgg
     1321 cggctgtcga ggcgcggcga gccgcagcca ttgcctttta tggtaatcgt gcgagagggc
     1381 gcagggactt cctttgtccc aaatctgtgc ggagccgaaa tctgggaggc gccgccgcac
     1441 cccctctagc gggcgcgggg cgaagcggtg cggcgccggc aggaaggaaa tgggcgggga
     1501 gggccttcgt gcgtcgccgc gccgccgtcc ccttctccct ctccagcctc ggggctgtcc
     1561 gcggggggac ggctgccttc gggggggacg gggcagggcg gggttcggct tctggcgtgt
     1621 gaccggcggc tctagagcct ctgctaacca tgttcatgcc ttcttctttt tcctacagct
     1681 cctgggcaac gtgctggtta ttgtgctgtc tcatcatttt ggcaaagaat tcgccaccat
     1741 ggctgccatc aggaagaaac tggtgattgt tggtgatgga gcttgtggta agacatgctt
     1801 gctcatagtc ttcagcaagg accagttccc agaggtctat gtgcccacgg tgtttgagaa
     1861 ttacgtagcc gacatagaag tggatgggaa gcaggtagag ttggctttat gggacacagc
     1921 tggacaggaa gattatgacc gcctgcggcc tctctcttat ccagacaccg atgttatatt
     1981 gatgtgtttt tccattgaca gtcccgactc acttgaaaac atcccagaaa aatggactcc
     2041 agaagtcaag catttctgtc caaatgtgcc catcatcctg gttgggaaca agaaggattt
     2101 cagaaacgat gaacataccc gacgggagtt ggccaaaatg aagcaggagc cggtaaaacc
     2161 tgaagaaggc agagatatgg caaacaggat tggcgctttt gggtacatgg agtgttcagc
     2221 aaagaccaaa gatggagtga gagaggtttt tgagatggcc acgagagctg ctctgcaagc
     2281 tagacgtggg aagaaaaagt ctgggtgcct catcttgtga gcggccgcac tcctcaggtg
     2341 caggctgcct atcagaaggt ggtggctggt gtggccaatg ccctggctca caaataccac
     2401 tgagatcttt ttccctctgc caaaaattat ggggacatca tgaagcccct tgagcatctg
     2461 acttctggct aataaaggaa atttattttc attgcaatag tgtgttggaa ttttttgtgt
     2521 ctctcactcg gaaggacata tgggagggca aatcatttaa aacatcagaa tgagtatttg
     2581 gtttagagtt tggcaacata tgcccatatg ctggctgcca tgaacaaagg ttggctataa
     2641 agaggtcatc agtatatgaa acagccccct gctgtccatt ccttattcca tagaaaagcc
     2701 ttgacttgag gttagatttt ttttatattt tgttttgtgt tatttttttc tttaacatcc
     2761 ctaaaatttt ccttacatgt tttactagcc agatttttcc tcctctcctg actactccca
     2821 gtcatagctg tccctcttct cttatggaga tccctcgacc tgcagcccaa gcttggcgta
     2881 atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat
     2941 acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt
     3001 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agcggatccg
     3061 catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact
     3121 ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag
     3181 gccgaggccg cctcggcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc
     3241 ctaggctttt gcaaaaagct aacttgttta ttgcagctta taatggttac aaataaagca
     3301 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt
     3361 ccaaactcat caatgtatct tatcatgtct ggatccgctg cattaatgaa tcggccaacg
     3421 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
     3481 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     3541 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     3601 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     3661 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     3721 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     3781 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     3841 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     3901 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     3961 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     4021 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     4081 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     4141 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     4201 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     4261 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     4321 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     4381 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     4441 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     4501 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     4561 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     4621 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     4681 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     4741 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     4801 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     4861 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     4921 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     4981 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     5041 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     5101 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     5161 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     5221 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     5281 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     5341 acaaataggg gttccgcgca catttccccg aaaagtgcca cctg
//
