LOCUS       sequence-349034-        6904 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  labelling of microtubule plus end and F-actin structures.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6904)
  AUTHORS   Alfadil E, Bradke F, Dupraz S
  TITLE     In Situ Visualization of Axon Growth and Growth Cone Dynamics in 
            Acute Ex Vivo Embryonic Brain Slice Cultures.
  JOURNAL   J Vis Exp. 2021 Oct 14;(176). doi: 10.3791/63068.
  PUBMED    34723948
REFERENCE   2  (bases 1 to 6904)
  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: "J Vis 
            Exp."; date: "2021-10-14"; volume: "(176). doi"; pages: " 
            10.3791/63068"
COMMENT     Sequence Label: pTub-alpha1-EB3-NeonGreen-T2A-iLifeact-mCherry
FEATURES             Location/Qualifiers
     source          1..6904
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      1267..1276
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      2125..2134
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2131..2838
                     /codon_start=1
                     /product="bright monomeric yellow-green fluorescent protein
                     derived from LanYFP (Shaner et al., 2013)"
                     /label=mNeonGreen
                     /translation="MVSKGEEDNMASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYE
                     ELNLKSTKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFED
                     GASLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIIS
                     TFKWSYTTGNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQ
                     KAFTDVMGMDELYK"
     regulatory      2386..2395
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2848..2901
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             2902..2949
                     /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="GVADLIKKFESISKEE"
     CDS             2974..3681
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(3116..3134)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3384..3403
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    3806..3927
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3843..3862)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3897..3916
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3934..4389)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4071..4092)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4283..4302
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4416..4520
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4522..4879
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4522..4713
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4545..4565)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4730..4865
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4914..5708
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             4914..5708
                     /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
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4968..4987)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5578..5597
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5896..5915)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5940..5987
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6316..6904
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6805..6824
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
       61 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgccat gcattagtta
      121 ttatgctgaa ttccgtatta ggagggatgg ctcatttcta gggacaaaat aacacaggct
      181 ctggggtgag gtagggtcgg gtaagggggc gagcggggag gggtgatggg agagcactac
      241 cgctaaagaa agcaagctct ctagtaaagc gttaagtact actttgtata ttgtttctct
      301 tttttccttt ctctctctct ctctctttgt ttgtttttgg tttttgagac aggatatcac
      361 actatatatc caggccgggt acaaaataac cgcagtcctc cgcttcccaa tgctgggatt
      421 tacaggcata aaccactaag ggcggttatg atgaccttga gctgcaagtc ttcctgcctc
      481 tgcctcccgg gtgctgttga gggtcatagg cgtggtctat tcatactgag cttctgaatt
      541 ttgctcaaat attaataata atagtaataa taataaccac aataatacaa ctgtaaaact
      601 aaacatttac ccacgccttt ttgaccatca ttcccatagc tcttgctact ttatttaaag
      661 cgaacagaga tgttgaatcc tgacggaacg tatttaaatt tagtgtagta taaatgaaaa
      721 gctggaattt accataaaga atctcaacac aaattctgtg attaagtgtt ggggaaaacc
      781 ttaaattatc ctaactacag tttaaggttt aagctccgta taatcaccca acccccgttt
      841 tctttcttcc ctctctaccc ctccccagct ccaccccata atggatgctc ggctagttgc
      901 ttttgcgagg cctttgtctg aaggatgcaa aatctacgga tgctagcgag ggggggaagg
      961 ggggagagat tacctcatac catgtcgctt gcaccaatca ccactctcct gtcgcggctt
     1021 ctctgggcag acggaggggt ctggaccaac aggaaaaggc cttggcccat ccccatggtg
     1081 accgagctgt atataaggag gcgcatccgc ccaagtgccg caggttctct tacatcgacc
     1141 gcttaagagt cgcgctgtaa gaagcaacac ctcctcctcg cctccgccat ccacccggca
     1201 gccgcgaagc agcaacaccg gtgagcgggg gatctcgagc tcaagcttcg aattctgcag
     1261 tcgactgcca ccatggccgt caatgtgtac tccacatctg tgaccagtga aaatctgagt
     1321 cgccatgata tgcttgcatg ggtcaacgac tccctgcacc tcaactatac caagatagaa
     1381 cagctttgtt caggggcagc ctactgccag ttcatggaca tgctcttccc cggctgtgtg
     1441 cacttgagga aagtgaagtt ccaggccaaa ctagagcatg aatacatcca caacttcaag
     1501 gtgctgcaag cagctttcaa gaagatgggt gttgacaaaa tcattcctgt agagaaatta
     1561 gtgaaaggaa aattccaaga taattttgag tttattcagt ggtttaagaa attctttgac
     1621 gcaaactatg atggaaagga ttacaaccct ctgctggcgc ggcagggcca ggacgtagcg
     1681 ccacctccta acccaggtga tcagatcttc aacaaatcca agaaactcat tggcacagca
     1741 gttccacaga ggacgtcccc cacaggccca aaaaacatgc agacctctgg ccggctgagc
     1801 aatgtggccc ccccctgcat tctccggaag aatcctccat cagcccgaaa tggcggccat
     1861 gagactgatg cccaaattct tgaactcaac caacagctgg tggacttgaa gctgacagtg
     1921 gatgggctgg agaaggaacg tgacttctac ttcagcaaac ttcgtgacat cgagctcatc
     1981 tgccaggagc atgaaagtga aaacagccct gttatctcag gcatcattgg catcctctat
     2041 gccacagagg aaggattcgc accccctgag gacgatgaga ttgaagagca tcaacaagaa
     2101 gaccaggacg agtacctgga tcccgccacc atggtgagca agggcgagga ggataacatg
     2161 gcctctctcc cagcgacaca tgagttacac atctttggct ccatcaacgg tgtggacttt
     2221 gacatggtgg gtcagggcac cggcaatcca aatgatggtt atgaggagtt aaacctgaag
     2281 tccaccaagg gtgacctcca gttctccccc tggattctgg tccctcatat cgggtatggc
     2341 ttccatcagt acctgcccta ccctgacggg atgtcgcctt tccaggccgc catggtagat
     2401 ggctccggct accaagtcca tcgcacaatg cagtttgaag atggtgcctc ccttactgtt
     2461 aactaccgct acacctacga gggaagccac atcaaaggag aggcccaggt gaaggggact
     2521 ggtttccctg ctgacggtcc tgtgatgacc aactcgctga ccgctgcgga ctggtgcagg
     2581 tcgaagaaga cttaccccaa cgacaaaacc atcatcagta cctttaagtg gagttacacc
     2641 actggaaatg gcaagcgcta ccggagcact gcgcggacca cctacacctt tgccaagcca
     2701 atggcggcta actatctgaa gaaccagccg atgtacgtgt tccgtaagac ggagctcaag
     2761 cactccaaga ccgagctcaa cttcaaggag tggcaaaagg cctttaccga tgtgatgggc
     2821 atggacgagc tgtacaaggg cagcggcgaa ggacgcggca gcctactgac ttgcggagat
     2881 gtcgaagaga accctggccc tggcgtggcc gacctgatca agaagttcga gagcatcagc
     2941 aaggaggagg gggatccacc ggtcgccacc ctggtgagca agggcgagga ggataacatg
     3001 gccatcatca aggagttcat gcgcttcaag gtgcacatgg agggctccgt gaacggccac
     3061 gagttcgaga tcgagggcga gggcgagggc cgcccctacg agggcaccca gaccgccaag
     3121 ctgaaggtga ccaagggtgg ccccctgccc ttcgcctggg acatcctgtc ccctcagttc
     3181 atgtacggct ccaaggccta cgtgaagcac cccgccgaca tccccgacta cttgaagctg
     3241 tccttccccg agggcttcaa gtgggagcgc gtgatgaact tcgaggacgg cggcgtggtg
     3301 accgtgaccc aggactcctc cctgcaggac ggcgagttca tctacaaggt gaagctgcgc
     3361 ggcaccaact tcccctccga cggccccgta atgcagaaga agaccatggg ctgggaggcc
     3421 tcctccgagc ggatgtaccc cgaggacggc gccctgaagg gcgagatcaa gcagaggctg
     3481 aagctgaagg acggcggcca ctacgacgct gaggtcaaga ccacctacaa ggccaagaag
     3541 cccgtgcagc tgcccggcgc ctacaacgtc aacatcaagt tggacatcac ctcccacaac
     3601 gaggactaca ccatcgtgga acagtacgaa cgcgccgagg gccgccactc caccggcggc
     3661 atggacgagc tgtacaagta atgagcggcc gcgactctag atcataatca gccataccac
     3721 atttgtagag gttttacttg ctttaaaaaa cctcccacac ctccccctga acctgaaaca
     3781 taaaatgaat gcaattgttg ttgttaactt gtttattgca gcttataatg gttacaaata
     3841 aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg
     3901 tttgtccaaa ctcatcaatg tatcttaagg cgtaaattgt aagcgttaat attttgttaa
     3961 aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca
     4021 aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga
     4081 acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc
     4141 agggcgatgg cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc
     4201 gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc
     4261 cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg
     4321 caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac
     4381 agggcgcgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     4441 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     4501 aatattgaaa aaggaagagt cctgaggcgg aaagaaccag ctgtggaatg tgtgtcagtt
     4561 agggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa
     4621 ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag
     4681 catgcatctc aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct
     4741 aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc
     4801 agaggccgag gccgcctcgg cctctgagct attccagaag tagtgaggag gcttttttgg
     4861 aggcctaggc ttttgcaaag atcgatcaag agacaggatg aggatcgttt cgcatgattg
     4921 aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg
     4981 actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg
     5041 ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaagacg
     5101 aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg
     5161 ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc
     5221 tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc
     5281 tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc
     5341 gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc
     5401 aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gagcatgccc gacggcgagg
     5461 atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct
     5521 tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt
     5581 tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc
     5641 tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt
     5701 tcttctgagc gggactctgg ggttcgaaat gaccgaccaa gcgacgccca acctgccatc
     5761 acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg
     5821 ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc
     5881 tagggggagg ctaactgaaa cacggaagga gacaataccg gaaggaaccc gcgctatgac
     5941 ggcaataaaa agacagaata aaacgcacgg tgttgggtcg tttgttcata aacgcggggt
     6001 tcggtcccag ggctggcact ctgtcgatac cccaccgaga ccccattggg gccaatacgc
     6061 ccgcgtttct tccttttccc caccccaccc cccaagttcg ggtgaaggcc cagggctcgc
     6121 agccaacgtc ggggcggcag gccctgccat agcctcaggt tactcatata tactttagat
     6181 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
     6241 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     6301 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     6361 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     6421 gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta
     6481 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     6541 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     6601 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     6661 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     6721 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     6781 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     6841 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     6901 gaaa
//
