LOCUS       sequence-243943-        7494 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Flp-dependent expression of tdTomato in mammalian cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7494)
  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 7494)
  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: pCAFNF-tdTomato
FEATURES             Location/Qualifiers
     source          1..7494
                     /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"
     protein_bind    1753..1786
                     /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             1829..2623
                     /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(1883..1902)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2493..2512
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    2803..2937
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    2946..2979
                     /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      3015..3024
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3021..4451
                     /codon_start=1
                     /product="tandem dimeric (pseudo-monomeric) derivative of 
                     DsRed (Shaner et al., 2004)"
                     /label=tdTomato
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVMNFEDGG
                     LVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGVLKGEIH
                     QALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYERSEGRH
                     HLFLGHGTGSTGSGSSGTASSEDNNMAVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPY
                     EGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVM
                     NFEDGGLVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGV
                     LKGEIHQALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYE
                     RSEGRHHLFLYGMDELYK"
     primer_bind     complement(4537..4556)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4602..4657
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4656..4675)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(5018..5034)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5018..5034)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5031..5053)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5042..5058
                     /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(5066..5072,5073..5090,5091..5096))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5111..5132
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      5217..5351
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    5371..5505
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5573..5590)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5744..6332)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5824..5843)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6503..7294,7295..7363))
                     /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     7126..7145
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7364..7468)
                     /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 gggggggcgc 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 tgattaattc
     1741 gagcgaacgc gtgaagttcc tattctctag aaagtatagg aacttccgga tttgatctga
     1801 tcaagagaca ggatgaggat cgtttcgcat gattgaacaa gatggattgc acgcaggttc
     1861 tccggccgct tgggtggaga ggctattcgg ctatgactgg gcacaacaga caatcggctg
     1921 ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac
     1981 cgacctgtcc ggtgccctga atgaactgca ggacgaggca gcgcggctat cgtggctggc
     2041 cacgacgggc gttccttgcg cagctgtgct cgacgttgtc actgaagcgg gaagggactg
     2101 gctgctattg ggcgaagtgc cggggcagga tctcctgtca tctcaccttg ctcctgccga
     2161 gaaagtatcc atcatggctg atgcaatgcg gcggctgcat acgcttgatc cggctacctg
     2221 cccattcgac caccaagcga aacatcgcat cgagcgagca cgtactcgga tggaagccgg
     2281 tcttgtcgat caggatgatc tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt
     2341 cgccaggctc aaggcgcgca tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc
     2401 ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct ggattcatcg actgtggccg
     2461 gctgggtgtg gcggaccgct atcaggacat agcgttggct acccgtgata ttgctgaaga
     2521 gcttggcggc gaatgggctg accgcttcct cgtgctttac ggtatcgccg ctcccgattc
     2581 gcagcgcatc gccttctatc gccttcttga cgagttcttc tgagcgggac tctggggttc
     2641 gaaatgaccg accaagcgac gcccaacctg ccatcacgag atttcgattc caccgccgcc
     2701 ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag
     2761 cgcggggatc tcatgctgga gttcttcgcc caccccatcg ataacttgtt tattgcagct
     2821 tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca
     2881 ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctggatcaaa
     2941 tccgagaagt tcctattctc tagaaagtat aggaacttca tctcgagtcg ctcggtacga
     3001 tttaaattga attcgccacc atggtgagca agggcgagga ggtcatcaaa gagttcatgc
     3061 gcttcaaggt gcgcatggag ggctccatga acggccacga gttcgagatc gagggcgagg
     3121 gcgagggccg cccctacgag ggcacccaga ccgccaagct gaaggtgacc aagggcggcc
     3181 ccctgccctt cgcctgggac atcctgtccc cccagttcat gtacggctcc aaggcgtacg
     3241 tgaagcaccc cgccgacatc cccgattaca agaagctgtc cttccccgag ggcttcaagt
     3301 gggagcgcgt gatgaacttc gaggacggcg gtctggtgac cgtgacccag gactcctccc
     3361 tgcaggacgg cacgctgatc tacaaggtga agatgcgcgg caccaacttc ccccccgacg
     3421 gccccgtaat gcagaagaag accatgggct gggaggcctc caccgagcgc ctgtaccccc
     3481 gcgacggcgt gctgaagggc gagatccacc aggccctgaa gctgaaggac ggcggccact
     3541 acctggtgga gttcaagacc atctacatgg ccaagaagcc cgtgcaactg cccggctact
     3601 actacgtgga caccaagctg gacatcacct cccacaacga ggactacacc atcgtggaac
     3661 agtacgagcg ctccgagggc cgccaccacc tgttcctggg gcatggcacc ggcagcaccg
     3721 gcagcggcag ctccggcacc gcctcctccg aggacaacaa catggccgtc atcaaagagt
     3781 tcatgcgctt caaggtgcgc atggagggct ccatgaacgg ccacgagttc gagatcgagg
     3841 gcgagggcga gggccgcccc tacgagggca cccagaccgc caagctgaag gtgaccaagg
     3901 gcggccccct gcccttcgcc tgggacatcc tgtcccccca gttcatgtac ggctccaagg
     3961 cgtacgtgaa gcaccccgcc gacatccccg attacaagaa gctgtccttc cccgagggct
     4021 tcaagtggga gcgcgtgatg aacttcgagg acggcggtct ggtgaccgtg acccaggact
     4081 cctccctgca ggacggcacg ctgatctaca aggtgaagat gcgcggcacc aacttccccc
     4141 ccgacggccc cgtaatgcag aagaagacca tgggctggga ggcctccacc gagcgcctgt
     4201 acccccgcga cggcgtgctg aagggcgaga tccaccaggc cctgaagctg aaggacggcg
     4261 gccactacct ggtggagttc aagaccatct acatggccaa gaagcccgtg caactgcccg
     4321 gctactacta cgtggacacc aagctggaca tcacctccca caacgaggac tacaccatcg
     4381 tggaacagta cgagcgctcc gagggccgcc accacctgtt cctgtacggc atggacgagc
     4441 tgtacaagta agcggccgca ctcctcaggt gcaggctgcc tatcagaagg tggtggctgg
     4501 tgtggccaat gccctggctc acaaatacca ctgagatctt tttccctctg ccaaaaatta
     4561 tggggacatc atgaagcccc ttgagcatct gacttctggc taataaagga aatttatttt
     4621 cattgcaata gtgtgttgga attttttgtg tctctcactc ggaaggacat atgggagggc
     4681 aaatcattta aaacatcaga atgagtattt ggtttagagt ttggcaacat atgcccatat
     4741 gctggctgcc atgaacaaag gttggctata aagaggtcat cagtatatga aacagccccc
     4801 tgctgtccat tccttattcc atagaaaagc cttgacttga ggttagattt tttttatatt
     4861 ttgttttgtg ttattttttt ctttaacatc cctaaaattt tccttacatg ttttactagc
     4921 cagatttttc ctcctctcct gactactccc agtcatagct gtccctcttc tcttatggag
     4981 atccctcgac ctgcagccca agcttggcgt aatcatggtc atagctgttt cctgtgtgaa
     5041 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
     5101 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     5161 agtcgggaaa cctgtcgtgc cagcggatcc gcatctcaat tagtcagcaa ccatagtccc
     5221 gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat
     5281 ttatgcagag gccgaggccg cctcggcctc tgagctattc cagaagtagt gaggaggctt
     5341 ttttggaggc ctaggctttt gcaaaaagct aacttgttta ttgcagctta taatggttac
     5401 aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt
     5461 tgtggtttgt ccaaactcat caatgtatct tatcatgtct ggatccgctg cattaatgaa
     5521 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca
     5581 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
     5641 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     5701 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     5761 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     5821 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     5881 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     5941 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     6001 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     6061 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     6121 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     6181 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     6241 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     6301 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     6361 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     6421 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     6481 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     6541 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     6601 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     6661 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     6721 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     6781 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     6841 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     6901 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     6961 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     7021 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     7081 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     7141 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     7201 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     7261 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     7321 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     7381 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     7441 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctg
//
