LOCUS       sequence-341013-        6853 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses LifeAct fused to EGFP Flp-dependently in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6853)
  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 6853)
  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-LifeAct-EGFP
FEATURES             Location/Qualifiers
     source          1..6853
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        5..384
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     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..3071
                     /codon_start=1
                     /product="peptide that binds filamentous actin (Riedl et 
                     al., 2008)"
                     /label=Lifeact
                     /note="first 17 amino acids of budding yeast Abp140"
                     /translation="MGVADLIKKFESISKEE"
     CDS             join(3093..3095,3096..3809)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(3093..3095,3096..3806)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(3135..3156)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3396..3415)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3743..3764
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3896..3915)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3961..4016
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4015..4034)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(4377..4393)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4377..4393)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4390..4412)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4401..4417
                     /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(4425..4431,4432..4449,4450..4455))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4470..4491
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      4576..4710
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    4730..4864
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4932..4949)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5103..5691)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5183..5202)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5862..6653,6654..6722))
                     /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     6485..6504
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6723..6827)
                     /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 gccrggsggg gsggggsggg gsgrggggsg
      541 gggsggggsg rggcggagag 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 atgggcgtgg ccgacttgat caagaagttc gagtccatct
     3061 ccaaggagga gggggatcca ccggtcgcca ccgtgagcaa gggcgaggag ctgttcaccg
     3121 gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt
     3181 ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca
     3241 ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt
     3301 gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg
     3361 aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg
     3421 ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact
     3481 tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac agccacaacg
     3541 tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag atccgccaca
     3601 acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg
     3661 acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccgcc ctgagcaaag
     3721 accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca
     3781 ctctcggcat ggacgagctg tacaagtaaa gcggccgcac tcctcaggtg caggctgcct
     3841 atcagaaggt ggtggctggt gtggccaatg ccctggctca caaataccac tgagatcttt
     3901 ttccctctgc caaaaattat ggggacatca tgaagcccct tgagcatctg acttctggct
     3961 aataaaggaa atttattttc attgcaatag tgtgttggaa ttttttgtgt ctctcactcg
     4021 gaaggacata tgggagggca aatcatttaa aacatcagaa tgagtatttg gtttagagtt
     4081 tggcaacata tgcccatatg ctggctgcca tgaacaaagg ttggctataa agaggtcatc
     4141 agtatatgaa acagccccct gctgtccatt ccttattcca tagaaaagcc ttgacttgag
     4201 gttagatttt ttttatattt tgttttgtgt tatttttttc tttaacatcc ctaaaatttt
     4261 ccttacatgt tttactagcc agatttttcc tcctctcctg actactccca gtcatagctg
     4321 tccctcttct cttatggaga tccctcgacc tgcagcccaa gcttggcgta atcatggtca
     4381 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
     4441 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg
     4501 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agcggatccg catctcaatt
     4561 agtcagcaac catagtcccg cccctaactc cgcccagttc cgcccattct ccgccccatg
     4621 gctgactaat tttttttatt tatgcagagg ccgaggccgc ctcggcctct gagctattcc
     4681 agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagcta acttgtttat
     4741 tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt
     4801 tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg
     4861 gatccgctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     4921 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     4981 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     5041 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     5101 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     5161 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     5221 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     5281 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     5341 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     5401 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     5461 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     5521 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     5581 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     5641 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     5701 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     5761 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     5821 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     5881 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     5941 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     6001 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     6061 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     6121 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     6181 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     6241 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     6301 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     6361 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     6421 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     6481 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     6541 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     6601 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     6661 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     6721 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     6781 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     6841 aaagtgccac ctg
//
