LOCUS       sequence-388077-        5798 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of the mApple-tagged N-terminal half of 
            photo-inactivated pi-EB1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5798)
  AUTHORS   Dema A, van Haren J, Wittmann T
  TITLE     Optogenetic EB1 inactivation shortens metaphase spindles by 
            disrupting cortical force-producing interactions with astral 
            microtubules.
  JOURNAL   Curr Biol. 2022 Mar 14;32(5):1197-1205.e4. doi: 
            10.1016/j.cub.2022.01.017. Epub 2022 Jan 31.
  PUBMED    35090591
REFERENCE   2  (bases 1 to 5798)
  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.cub.2022.01.017"; journalName: "Curr Biol"; date: 
            "2022-03-14- 14"; volume: "32"; issue: "5"; pages: "1197-1205.e4"
COMMENT     Sequence Label: pEB1N-mApple-LZ-LOV2
FEATURES             Location/Qualifiers
     source          1..5798
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        61..364
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     519..539
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             1161..1868
                     /codon_start=1
                     /product="photostable monomeric derivative of DsRed (Shaner
                     et al., 2008)"
                     /label=mApple
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEENNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEA
                     FQTAKLKVTKGGPLPFAWDILSPQFMYGSKVYIKHPADIPDYFKLSFPEGFRWERVMNF
                     EDGGIIHVNQDSSLQDGVFIYKVKLRGTNFPSDGPVMQKKTMGWEASEERMYPEDGALK
                     SEIKKRLKLKDGGHYAAEVKTTYKAKKPVQLPGAYIVDIKLDIVSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     1574..1593
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             1899..1955
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility 
                     while preserving binding to an scFv-GFP fusion protein 
                     (Tanenbaum et al., 2014)"
                     /label=GCN4_v4
                     /translation="EELLSKNYHLENEVARLKK"
     polyA_signal    2586..2707
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2623..2642)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2677..2696
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2714..3169)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2851..2872)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3063..3082
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3196..3300
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3302..3659
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3302..3493
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3325..3345)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3510..3645
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3694..4488
                     /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             3694..4488
                     /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(3748..3767)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4358..4377
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4676..4695)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4720..4767
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5096..5684
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5585..5604
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcacca
      601 tggcagtgaa cgtatactca acgtcagtga ccagtgataa cctaagtcga catgacatgc
      661 tggcctggat caatgagtct ctgcagttga atctgacaaa gatcgaacag ttgtgctcag
      721 gggctgcgta ttgtcagttt atggacatgc tgttccctgg ctccattgcc ttgaagaaag
      781 tgaaattcca agctaagcta gaacacgagt acatccagaa cttcaaaata ttacaagcag
      841 gttttaagag aatgggtgtt gacaaaataa tcccggtgga caaattagta aaaggaaagt
      901 ttcaggacaa ttttgaattc gttcagtggt tcaagaagtt cttcgatgca aactatgatg
      961 gaaaagacta tgaccctgtg gctgctagac aaggtcaaga aactgcagtg gctccttccc
     1021 ttgttgctcc agctctgaat aaaccgaaga aacctctcac ttctagcagt gcagctcccc
     1081 agaggcccat ctcaacacag agaaccgctg cggctcctaa ggctggccct ggtgtggtgc
     1141 gaaagaaccc tggtctcgag atggtgagca agggcgagga gaataacatg gccatcatca
     1201 aggagttcat gcgcttcaag gtgcacatgg agggctccgt gaacggccac gagttcgaga
     1261 tcgagggcga gggcgagggc cgcccctacg aggcctttca gaccgctaag ctgaaggtga
     1321 ccaagggtgg ccccctgccc ttcgcctggg acatcctgtc ccctcagttc atgtacggct
     1381 ccaaggtcta cattaagcac ccagccgaca tccccgacta cttcaagctg tccttccccg
     1441 agggcttcag gtgggagcgc gtgatgaact tcgaggacgg cggcattatt cacgttaacc
     1501 aggactcctc cctgcaggac ggcgtgttca tctacaaggt gaagctgcgc ggcaccaact
     1561 tcccctccga cggccccgta atgcagaaga agaccatggg ctgggaggcc tccgaggagc
     1621 ggatgtaccc cgaggacggc gccctgaaga gcgagatcaa gaagaggctg aagctgaagg
     1681 acggcggcca ctacgccgcc gaggtcaaga ccacctacaa ggccaagaag cccgtgcagc
     1741 tgcccggcgc ctacatcgtc gacatcaagt tggacatcgt gtcccacaac gaggactaca
     1801 ccatcgtgga acagtacgaa cgcgccgagg gccgccactc caccggcggc atggacgagc
     1861 tgtacaaggg aggtatgaaa cagctggaag ataaagtgga agagctgttg agcaaaaact
     1921 atcatctgga aaacgaagtg gcgcgtctga aaaagctggt gggcgaactc gagctcaagc
     1981 ttcgaattct gcagtcgacg gtaccgcggg gttctggaag tggattggct gctgcacttg
     2041 aacgtattga gaagaacttt gtcattactg acccaagatt gccagataat cccattatat
     2101 tcgcgtccga tagtttcttg cagttgacag aatatagccg tgaagaaatt ttgggaagaa
     2161 actgcaggtt tctacaaggt cctgaaactg atcgcgcgac agtgagaaaa attagagatg
     2221 ccatagataa ccaaacagag gtcactgttc agctgattaa ttatacaaag agtggtaaaa
     2281 agttctggaa cctctttcac ttgcagccta tgcgagatca gaagggagat gtccagtact
     2341 ttattggggt tcagttggat ggaactgagc atgtccgaga tgctgccgag agagagggag
     2401 tcatgctgat taagaaaact gcagaaaata ttgatgaggc ggcaaaagaa ctttaaggat
     2461 ccaccggatc tagataactg atcataatca gccataccac atttgtagag gttttacttg
     2521 ctttaaaaaa cctcccacac ctccccctga acctgaaaca taaaatgaat gcaattgttg
     2581 ttgttaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt
     2641 tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg
     2701 tatcttaacg cgtaaattgt aagcgttaat attttgttaa aattcgcgtt aaatttttgt
     2761 taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa
     2821 gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag
     2881 aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt
     2941 gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac
     3001 cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag
     3061 gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg
     3121 cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc aggtggcact
     3181 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     3241 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
     3301 cctgaggcgg aaagaaccag ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag
     3361 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accaggtgtg
     3421 gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag
     3481 caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc
     3541 attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctcgg
     3601 cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaag
     3661 atcgatcaag agacaggatg aggatcgttt cgcatgattg aacaagatgg attgcacgca
     3721 ggttctccgg ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacaatc
     3781 ggctgctctg atgccgccgt gttccggctg tcagcgcagg ggcgcccggt tctttttgtc
     3841 aagaccgacc tgtccggtgc cctgaatgaa ctgcaagacg aggcagcgcg gctatcgtgg
     3901 ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg
     3961 gactggctgc tattgggcga agtgccgggg caggatctcc tgtcatctca ccttgctcct
     4021 gccgagaaag tatccatcat ggctgatgca atgcggcggc tgcatacgct tgatccggct
     4081 acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac tcggatggaa
     4141 gccggtcttg tcgatcagga tgatctggac gaagagcatc aggggctcgc gccagccgaa
     4201 ctgttcgcca ggctcaaggc gagcatgccc gacggcgagg atctcgtcgt gacccatggc
     4261 gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt
     4321 ggccggctgg gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct
     4381 gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc
     4441 gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc gggactctgg
     4501 ggttcgaaat gaccgaccaa gcgacgccca acctgccatc acgagatttc gattccaccg
     4561 ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc
     4621 tccagcgcgg ggatctcatg ctggagttct tcgcccaccc tagggggagg ctaactgaaa
     4681 cacggaagga gacaataccg gaaggaaccc gcgctatgac ggcaataaaa agacagaata
     4741 aaacgcacgg tgttgggtcg tttgttcata aacgcggggt tcggtcccag ggctggcact
     4801 ctgtcgatac cccaccgaga ccccattggg gccaatacgc ccgcgtttct tccttttccc
     4861 caccccaccc cccaagttcg ggtgaaggcc cagggctcgc agccaacgtc ggggcggcag
     4921 gccctgccat agcctcaggt tactcatata tactttagat tgatttaaaa cttcattttt
     4981 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     5041 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     5101 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     5161 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     5221 gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga
     5281 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     5341 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     5401 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     5461 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     5521 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     5581 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     5641 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     5701 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     5761 cccctgattc tgtggataac cgtattaccg ccatgcat
//
