LOCUS       sequence-401323-        6102 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mEGFP-tagged tardigrade (Hypsibius exemplaris) epsilon-tubulin Tub5 
            for expression in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6102)
  AUTHORS   Novotna Floriancicova K, Baltzis A, Smejkal J, Czernekova M, 
            Kaczmarek L, Maly J, Notredame C, Vinopal S
  TITLE     Phylogenetic and functional characterization of water bears 
            (Tardigrada) tubulins.
  JOURNAL   Sci Rep. 2023 Mar 30;13(1):5194. doi: 10.1038/s41598-023-31992-z.
  PUBMED    36997657
REFERENCE   2  (bases 1 to 6102)
  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: "Sci Rep."; 
            date: "2023-03-30"; volume: "13(1):5194. doi"; pages: " 
            10.1038/s41598-023-31992-z"
COMMENT     Sequence Label: pmEGFP-tHe-Tub5
FEATURES             Location/Qualifiers
     source          1..6102
                     /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"
     regulatory      595..604
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(601..603,604..606,607..1317)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLK
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(646..667)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(907..926)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     polyA_signal    2890..3011
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2927..2946)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2981..3000
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3018..3473)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3155..3176)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3367..3386
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3500..3604
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3606..3963
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3606..3797
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3629..3649)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3814..3949
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3998..4792
                     /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             3998..4792
                     /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(4052..4071)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4662..4681
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4980..4999)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5024..5071
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5400..5988
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5889..5908
                     /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 gctagccacc
      601 atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac
      661 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac
      721 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc
      781 ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag
      841 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc
      901 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg
      961 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac
     1021 aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac
     1081 ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc
     1141 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac
     1201 tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc
     1261 cttaaggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtcc
     1321 ggactcagat ctcgagtgtc gcataatgtg gtcatctcga tcgggcagtg cggcaatcaa
     1381 attggatcag agttttggga tcgcgtcgtc cattcacacg ccttactgga caccgatggg
     1441 actttcactt cagcgcttgc cacttttttc cgcaacaccg atggccgaca cgattcgagt
     1501 ctacctatcg gtagtcgact caagcatttg caggccaacg ccgtgcttat cgacatggaa
     1561 actcgcgtgg tggacaggct cctccgcggc ccgttgggtg atctgtttga caagcggatg
     1621 acgttgacag actcctgtgg aagcgggaac aactggtcca ctggacatct gacctacggc
     1681 ctgcaacatg aagacacgat ttcggagatg ctccggagaa gtgcagagcc gcttgaccag
     1741 ctgcagtctt tttcagtcct tcactctatg ggcggtggaa ctggcagtgg cttgggtacg
     1801 gccgtggtga agatgatggc acgggattat ggcaaaacgg cgcggttggt cagttgcgtt
     1861 tttcccgggg atcaggatga tgtcgtcacg tcgccgtaca attctgttct ggctctgaag
     1921 gtattggctg aagacgctga tagcgtgatt agcctcgaca acaacgccct ctccacagtc
     1981 tgttcaaaac acgttctcgc cggccggcca cttcgaaaaa gtcctcccgt tccgttcgat
     2041 caaatgaacg acttggtagc ccggatgttg gccgacctga ccagttccat tcgcttcggc
     2101 ggcgaactca gtattccgct gagcgagctc cccgtccgga tgagcccgtt cccgcggatg
     2161 aaattcctca tacccggatt gagtcccgtg actggagcgc tgcccaatat ggagtcgaag
     2221 aagattgacc ggaccttcta cgaagcgttc gagcgacgca atcaactgat ggactgcgac
     2281 ccgcagaggt ccgcgtacct tgcctgtctc ctactcgctc gtgggaaaga catcgcgccg
     2341 tcggacgtgc aacggaacgt cgataagatg aagcggtaca tccacttcgc gccgggcagt
     2401 aaggacgagt gggcggtctc actgtgcggg attccctcgc agtcgcatcc acggtccttc
     2461 ttgacgctgg ccaacaacgg ctgcttccac aagccgatgc agtgcctgaa ggagcgattt
     2521 gtcgccatgt accgtgggcg agcacacctc catcactacc tgctgaacgg gatggaggcg
     2581 gccgatttcg aggccagctt ggagagcttg tcagcgttga tcagcgacta caggcaagtt
     2641 gatgctgccg gcgctgtcgc agccaatgtg cgggatcgtg gatacgtcgc cgtcggcgtg
     2701 gtgaagccca aggtgaaggc gggctgatcg aattctgcag tcgacggtac cgcgggcccg
     2761 ggatccaccg gatctagata actgatcata atcagccata ccacatttgt agaggtttta
     2821 cttgctttaa aaaacctccc acacctcccc ctgaacctga aacataaaat gaatgcaatt
     2881 gttgttgtta acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
     2941 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
     3001 aatgtatctt aacgcgtaaa ttgtaagcgt taatattttg ttaaaattcg cgttaaattt
     3061 ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc
     3121 aaaagaatag accgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt
     3181 aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact
     3241 acgtgaacca tcaccctaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg
     3301 gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag
     3361 aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac
     3421 gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtcaggtgg
     3481 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     3541 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
     3601 gagtcctgag gcggaaagaa ccagctgtgg aatgtgtgtc agttagggtg tggaaagtcc
     3661 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccagg
     3721 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag
     3781 tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc
     3841 gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc
     3901 tcggcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc
     3961 aaagatcgat caagagacag gatgaggatc gtttcgcatg attgaacaag atggattgca
     4021 cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac
     4081 aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt
     4141 tgtcaagacc gacctgtccg gtgccctgaa tgaactgcaa gacgaggcag cgcggctatc
     4201 gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg
     4261 aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc
     4321 tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc
     4381 ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat
     4441 ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc
     4501 cgaactgttc gccaggctca aggcgagcat gcccgacggc gaggatctcg tcgtgaccca
     4561 tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga
     4621 ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat
     4681 tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc
     4741 tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct gagcgggact
     4801 ctggggttcg aaatgaccga ccaagcgacg cccaacctgc catcacgaga tttcgattcc
     4861 accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg
     4921 atcctccagc gcggggatct catgctggag ttcttcgccc accctagggg gaggctaact
     4981 gaaacacgga aggagacaat accggaagga acccgcgcta tgacggcaat aaaaagacag
     5041 aataaaacgc acggtgttgg gtcgtttgtt cataaacgcg gggttcggtc ccagggctgg
     5101 cactctgtcg ataccccacc gagaccccat tggggccaat acgcccgcgt ttcttccttt
     5161 tccccacccc accccccaag ttcgggtgaa ggcccagggc tcgcagccaa cgtcggggcg
     5221 gcaggccctg ccatagcctc aggttactca tatatacttt agattgattt aaaacttcat
     5281 ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct
     5341 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     5401 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     5461 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     5521 agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc
     5581 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     5641 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     5701 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     5761 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     5821 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     5881 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     5941 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     6001 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     6061 ttatcccctg attctgtgga taaccgtatt accgccatgc at
//
