LOCUS       sequence-341076-        6646 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses RhoA Flp-dependently in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6646)
  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 6646)
  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: pCAFNF-Rhoa
FEATURES             Location/Qualifiers
     source          1..6646
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        5..384
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        386..662
                     /label=chicken beta-actin promoter
     intron          663..1679
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1603..1625
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1687..1706
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     protein_bind    1754..1787
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     CDS             1830..2624
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(1884..1903)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2494..2513
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    2804..2938
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    2947..2980
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     regulatory      3016..3025
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(3689..3708)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3754..3809
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3808..3827)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(4170..4186)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4170..4186)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4183..4205)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4194..4210
                     /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(4218..4224,4225..4242,4243..4248))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4263..4284
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      4369..4503
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    4523..4657
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4725..4742)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4896..5484)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4976..4995)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5655..6446,6447..6515))
                     /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     6278..6297
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6516..6620)
                     /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 gggggnsgcg cgccrggsgg ggsggggsgg ggsgrggggs
      541 ggggsggggc gaggcggaga ggtgcggcgg cagccaatca gagcggcgcg ctccgaaagt
      601 ttccttttat ggcgaggcgg cggcggcggc ggccctataa aaagcgaagc gcgcggcggg
      661 cgggagtcgc tgcgcgctgc cttcgccccg tgccccgctc cgccgccgcc tcgcgccgcc
      721 cgccccggct ctgactgacc gcgttactcc cacaggtgag cgggcgggac ggcccttctc
      781 ctccgggctg taattagcgc ttggtttaat gacggcttgt ttcttttctg tggctgcgtg
      841 aaagccttga ggggctccgg gagggccctt tgtgcggggg gagcggctcg gggggtgcgt
      901 gcgtgtgtgt gtgcgtgggg agcgccgcgt gcggctccgc gctgcccggc ggctgtgagc
      961 gctgcgggcg cggcgcgggg ctttgtgcgc tccgcagtgt gcgcgagggg agcgcggccg
     1021 ggggcggtgc cccgcggtgc ggggggggct gcgaggggaa caaaggctgc gtgcggggtg
     1081 tgtgcgtggg ggggtgagca gggggtgtgg gcgcgtcggt cgggctgcaa ccccccctgc
     1141 acccccctcc ccgagttgct gagcacggcc cggcttcggg tgcggggctc cgtacggggc
     1201 gtggcgcggg gctcgccgtg ccgggcgggg ggtggcggca ggtgggggtg ccgggcgggg
     1261 cggggccgcc tcgggccggg gagggctcgg gggaggggcg cggcggcccc cggagcgccg
     1321 gcggctgtcg aggcgcggcg agccgcagcc attgcctttt atggtaatcg tgcgagaggg
     1381 cgcagggact tcctttgtcc caaatctgtg cggagccgaa atctgggagg cgccgccgca
     1441 ccccctctag cgggcgcggg gcgaagcggt gcggcgccgg caggaaggaa atgggcgggg
     1501 agggccttcg tgcgtcgccg cgccgccgtc cccttctccc tctccagcct cggggctgtc
     1561 cgcgggggga cggctgcctt cgggggggac ggggcagggc ggggttcggc ttctggcgtg
     1621 tgaccggcgg ctctagagcc tctgctaacc atgttcatgc cttcttcttt ttcctacagc
     1681 tcctgggcaa cgtgctggtt attgtgctgt ctcatcattt tggcaaagaa ttgattaatt
     1741 cgagcgaacg cgtgaagttc ctattctcta gaaagtatag gaacttccgg atttgatctg
     1801 atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg cacgcaggtt
     1861 ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag acaatcggct
     1921 gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt tttgtcaaga
     1981 ccgacctgtc cggtgccctg aatgaactgc aggacgaggc agcgcggcta tcgtggctgg
     2041 ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg ggaagggact
     2101 ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt gctcctgccg
     2161 agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat ccggctacct
     2221 gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg atggaagccg
     2281 gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca gccgaactgt
     2341 tcgccaggct caaggcgcgc atgcccgacg gcgaggatct cgtcgtgacc catggcgatg
     2401 cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc gactgtggcc
     2461 ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat attgctgaag
     2521 agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt
     2581 cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgagcggga ctctggggtt
     2641 cgaaatgacc gaccaagcga cgcccaacct gccatcacga gatttcgatt ccaccgccgc
     2701 cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca
     2761 gcgcggggat ctcatgctgg agttcttcgc ccaccccatc gataacttgt ttattgcagc
     2821 ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc
     2881 actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg tctggatcaa
     2941 atccgagaag ttcctattct ctagaaagta taggaacttc atctcgagtc gctcggtacg
     3001 atttaaattg aattcgccac catggctgcc atcaggaaga aactggtgat tgttggtgat
     3061 ggagcttgtg gtaagacatg cttgctcata gtcttcagca aggaccagtt cccagaggtc
     3121 tatgtgccca cggtgtttga aaactatgtg gcggatatcg aggtggatgg gaagcaggta
     3181 gagttggctt tatgggacac agctggacag gaagattatg accgcctgcg gcctctctct
     3241 tatccagaca ccgatgttat attgatgtgt ttttccattg acagccctga tagtttagaa
     3301 aacatcccag aaaaatggac tccagaagtc aagcatttct gtccaaatgt gcccatcatc
     3361 ctggttggga acaagaagga ccttcggaat gacgagcaca cgagacggga gttggccaaa
     3421 atgaagcagg agccggtaaa acctgaagaa ggcagagata tggcaaacag gattggcgct
     3481 tttgggtaca tggagtgttc agcaaagacc aaagatggag tgagagaggt ttttgagatg
     3541 gccacgagag ctgctctgca agctagacgt gggaagaaaa agtctgggtg cctcatcttg
     3601 tgagcggccg cactcctcag gtgcaggctg cctatcagaa ggtggtggct ggtgtggcca
     3661 atgccctggc tcacaaatac cactgagatc tttttccctc tgccaaaaat tatggggaca
     3721 tcatgaagcc ccttgagcat ctgacttctg gctaataaag gaaatttatt ttcattgcaa
     3781 tagtgtgttg gaattttttg tgtctctcac tcggaaggac atatgggagg gcaaatcatt
     3841 taaaacatca gaatgagtat ttggtttaga gtttggcaac atatgcccat atgctggctg
     3901 ccatgaacaa aggttggcta taaagaggtc atcagtatat gaaacagccc cctgctgtcc
     3961 attccttatt ccatagaaaa gccttgactt gaggttagat tttttttata ttttgttttg
     4021 tgttattttt ttctttaaca tccctaaaat tttccttaca tgttttacta gccagatttt
     4081 tcctcctctc ctgactactc ccagtcatag ctgtccctct tctcttatgg agatccctcg
     4141 acctgcagcc caagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat
     4201 ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc
     4261 taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     4321 aacctgtcgt gccagcggat ccgcatctca attagtcagc aaccatagtc ccgcccctaa
     4381 ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag
     4441 aggccgaggc cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag
     4501 gcctaggctt ttgcaaaaag ctaacttgtt tattgcagct tataatggtt acaaataaag
     4561 caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt
     4621 gtccaaactc atcaatgtat cttatcatgt ctggatccgc tgcattaatg aatcggccaa
     4681 cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg
     4741 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
     4801 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
     4861 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     4921 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     4981 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     5041 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     5101 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     5161 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     5221 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     5281 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca
     5341 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     5401 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     5461 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     5521 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     5581 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     5641 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     5701 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     5761 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
     5821 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     5881 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     5941 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
     6001 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     6061 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     6121 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     6181 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     6241 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
     6301 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     6361 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     6421 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     6481 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
     6541 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     6601 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctg
//
