LOCUS       sequence-401316-        6060 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mEGFP-tagged tardigrade (Hypsibius exemplaris) alpha-tubulin Tub1A2 
            for expression in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6060)
  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 6060)
  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-Tub1A2
FEATURES             Location/Qualifiers
     source          1..6060
                     /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    2848..2969
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2885..2904)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2939..2958
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2976..3431)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3113..3134)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3325..3344
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3458..3562
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3564..3921
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3564..3755
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3587..3607)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3772..3907
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3956..4750
                     /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             3956..4750
                     /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(4010..4029)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4620..4639
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4938..4957)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4982..5029
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5358..5946
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5847..5866
                     /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 ctcgagtgcg cgaatgcatc tctatccacg tcggacaggc cggagtgcag
     1381 attggcaacg cctgctggga gctttactgt ctggagcatg gcatccagcc ggacgggcag
     1441 atgccgtcgg acaagacggt tggcgccgct gacgactcct tcaacacctt cttcagcgag
     1501 accggatccg gcaagcacgt tccgcgggcc gtcttcgtcg atctcgagcc gaccgttgtt
     1561 gatgaagtgc gaaccggaac ataccgccag cttttccatc cagagcagct aatcaccgga
     1621 aaggaggatg ccgccaacaa ttacgctcgc gggcactaca ccgtcggcaa ggagattgtc
     1681 gacctggtgc tggaccgcgt gcgcaagctc gccgaccaat gtaccggtct gcagggcttt
     1741 ctcatctttc actcgttcgg cggtgggacc ggatccggat tcacctccct gctgatggaa
     1801 cgcttgtcgg tcgactacgg gaagaagtcc aaactggaat tctccatcta ccccgcccct
     1861 caggtgtcga ctgccgtggt ggagccgtac aactcgatcc tgacgaccca taccacgctg
     1921 gagcactctg actgtgcctt catggtgaac aacgaagcca tctacgacat ttgccgtcgg
     1981 aaccttgaca ttgagcgtcc gacctacacc aacctcaacc gcctgatcgg gcagattgtc
     2041 tcctccatca ccgcgtcgct gcgcttcgac ggcgctctca acgtcgatct caccgaattc
     2101 cagaccaact tggttcctta cccccgtatc catttcccgc tggtgaccta cgccccgatc
     2161 atttccgctg aaaaggccta tcacgaacag ttgacggtct cggagatcac caactcgtgc
     2221 ttcgagccgg ctaaccagat ggtcaagtgc gacccgcgcc acggaaagta catggcctgc
     2281 tgcatgctct accgcggtga cgttgtgccc aaggacgtga acgccgccat cgcggccatt
     2341 aagaccaagc gcagcatcca gtttgtcgac tggtgcccga ctggatttaa ggtgggaatc
     2401 aactatcagc ccccgacggt ggtgccgggc ggggatttgg ccaaggtgca gcgtgccgtc
     2461 tgcatgctgt ccaacaccac ggccatcgcc gaagcctggg cgcgtctgga ccacaagttc
     2521 gacctgatgt acgccaagcg cgcctttgtc cactggtacg tcggtgaggg catggaggag
     2581 ggcgagttca gcgaggcccg tgaagatttg gccgccctcg agaaggacta cgaggaggtc
     2641 ggtgtggact ctgtcgacgc tggtgaggaa ggcgaggaat attgatcgaa ttctgcagtc
     2701 gacggtaccg cgggcccggg atccaccgga tctagataac tgatcataat cagccatacc
     2761 acatttgtag aggttttact tgctttaaaa aacctcccac acctccccct gaacctgaaa
     2821 cataaaatga atgcaattgt tgttgttaac ttgtttattg cagcttataa tggttacaaa
     2881 taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt
     2941 ggtttgtcca aactcatcaa tgtatcttaa cgcgtaaatt gtaagcgtta atattttgtt
     3001 aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg
     3061 caaaatccct tataaatcaa aagaatagac cgagataggg ttgagtgttg ttccagtttg
     3121 gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta
     3181 tcagggcgat ggcccactac gtgaaccatc accctaatca agttttttgg ggtcgaggtg
     3241 ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa
     3301 gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct
     3361 ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta atgcgccgct
     3421 acagggcgcg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
     3481 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca
     3541 ataatattga aaaaggaaga gtcctgaggc ggaaagaacc agctgtggaa tgtgtgtcag
     3601 ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     3661 aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
     3721 agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc
     3781 ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat
     3841 gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgagg aggctttttt
     3901 ggaggcctag gcttttgcaa agatcgatca agagacagga tgaggatcgt ttcgcatgat
     3961 tgaacaagat ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta
     4021 tgactgggca caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca
     4081 ggggcgcccg gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcaaga
     4141 cgaggcagcg cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga
     4201 cgttgtcact gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct
     4261 cctgtcatct caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg
     4321 gctgcatacg cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga
     4381 gcgagcacgt actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca
     4441 tcaggggctc gcgccagccg aactgttcgc caggctcaag gcgagcatgc ccgacggcga
     4501 ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg
     4561 cttttctgga ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc
     4621 gttggctacc cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt
     4681 gctttacggt atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga
     4741 gttcttctga gcgggactct ggggttcgaa atgaccgacc aagcgacgcc caacctgcca
     4801 tcacgagatt tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc
     4861 cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac
     4921 cctaggggga ggctaactga aacacggaag gagacaatac cggaaggaac ccgcgctatg
     4981 acggcaataa aaagacagaa taaaacgcac ggtgttgggt cgtttgttca taaacgcggg
     5041 gttcggtccc agggctggca ctctgtcgat accccaccga gaccccattg gggccaatac
     5101 gcccgcgttt cttccttttc cccaccccac cccccaagtt cgggtgaagg cccagggctc
     5161 gcagccaacg tcggggcggc aggccctgcc atagcctcag gttactcata tatactttag
     5221 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat
     5281 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     5341 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     5401 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     5461 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg
     5521 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     5581 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     5641 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     5701 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     5761 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     5821 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     5881 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     5941 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     6001 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgccatgcat
//
