LOCUS       sequence-401321-        6010 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tardigrade (Hypsibius exemplaris) beta-tubulin Tub2D4 tagged with 
            mScarlet for expression in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6010)
  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 6010)
  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: ptHe-Tub2D4-mScarlet
FEATURES             Location/Qualifiers
     source          1..6010
                     /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      1971..1980
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1977..2672
                     /codon_start=1
                     /product="bright monomeric red fluorescent protein evolved 
                     from a synthetic template (Bindels et al., 2016)"
                     /label=mScarlet
                     /translation="MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFSWDILSPQFMYGSRAFTKHPADIPDYYKQSFPEGFKWERVMNFEDGG
                     AVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIK
                     MALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRH
                     STGGMDELYK"
     primer_bind     complement(2110..2128)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2378..2397
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    2798..2919
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2835..2854)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2889..2908
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2926..3381)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3063..3084)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3275..3294
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3408..3512
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3514..3871
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3514..3705
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3537..3557)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3722..3857
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3906..4700
                     /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             3906..4700
                     /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(3960..3979)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4570..4589
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4888..4907)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4932..4979
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5308..5896
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5797..5816
                     /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 gctagcgcca
      601 ccatgcgcga aatcgtccac ctccaagtcg gccagtgcgg caaccagatc ggagcgaagt
      661 tctgggaagt gatctccgac gaacatggca tcgaccccac cggcacctac cgcggcgact
      721 cggatttgca gttggaacgg atcaatgttt actataacga agctggaaac aacaaatatg
      781 ttccgcgcgc tgttttggtc gatcttgagc cgggaacgat ggattcggtc cgatcatctc
      841 cgtttggtca acttttccga ccggataact ttatctttgg ccagagtggt gcgggaaaca
      901 actgggctaa aggccactac accgagggtg ccgaactggt ggactctgtt ctcgatgtcg
      961 tgcgcaagga ggccgagaac tgcgactgcc tccagggatt tcagctgact cactcgctgg
     1021 gcggcggcac tggatccggt atgggtactc tgctcatctc taagatccgc gaggagtacc
     1081 ccgatcgcat catgaccaca ttcagcgttg ttccatcgcc gaaggtgtct gacactgtgg
     1141 tggagcccta caacgcgact ctgtcggtcc accagctggt cgagaatacc gacgagacgt
     1201 tttgcatcga caacgaagcg ctgtacgaca tctgcttccg caccctcaag ctgaccaccc
     1261 cgacgtacgg cgacctgaac cacctcgtgt ctgccaccat gtcgggtgtg acgacctgct
     1321 tccgtttccc gggccaactg aacgccgatc tccgcaagtt ggccgtcaac atggtgccct
     1381 tcccgcggct gcactttttc atgcccggtt tcgccccgtt gacagctcgc ggcagccagg
     1441 gctaccgtgc cctcagcgtc gccgaactca ccacccagat gttcgactcg aagaacatga
     1501 tggccgcctg cgatccccgt cacggacgct acctcaccgt ggcggccatc ttccgcggcc
     1561 ggatgagcat gaaggaagtg gacgaccaaa tgctgcagat ccagaacaag aactccagct
     1621 acttcgttga atggattccc aacaacatca agacagccgt ctgcgacatc cctccgcgcg
     1681 gtctgaagat ggcggccacg ttcatcggca acagcactgc catccaggag ctgttcaagc
     1741 gcatcagcga gcagttcact gccatgttcc gtcgcaaggc cttcctccat tggtacaccg
     1801 gcgagggcat ggacgagatg gaattcaccg aggccgagag caacatgaac gatctcatct
     1861 cggagtacca gcagtaccag gaggccaccg ccgaggatga gggtgaattt gaggagggcg
     1921 agcaggaggc ccaggaggag gctgctggag gtggtggagg ttcaccggtc gccaccatgg
     1981 tgagcaaggg cgaggcagtg atcaaggagt tcatgcggtt caaggtgcac atggagggct
     2041 ccatgaacgg ccacgagttc gagatcgagg gcgagggcga gggccgcccc tacgagggca
     2101 cccagaccgc caagctgaag gtgaccaagg gtggccccct gcccttctcc tgggacatcc
     2161 tgtcccctca gttcatgtac ggctccaggg ccttcaccaa gcaccccgcc gacatccccg
     2221 actactataa gcagtccttc cccgagggct tcaagtggga gcgcgtgatg aacttcgagg
     2281 acggcggcgc cgtgaccgtg acccaggaca cctccctgga ggacggcacc ctgatctaca
     2341 aggtgaagct ccgcggcacc aacttccctc ctgacggccc cgtaatgcag aagaagacaa
     2401 tgggctggga agcgtccacc gagcggttgt accccgagga cggcgtgctg aagggcgaca
     2461 ttaagatggc cctgcgcctg aaggacggcg gccgctacct ggcggacttc aagaccacct
     2521 acaaggccaa gaagcccgtg cagatgcccg gcgcctacaa cgtcgaccgc aagttggaca
     2581 tcacctccca caacgaggac tacaccgtgg tggaacagta cgaacgctcc gagggccgcc
     2641 actccaccgg cggcatggac gagctgtaca agtaaagcgg ccgcgactct agatcataat
     2701 cagccatacc acatttgtag aggttttact tgctttaaaa aacctcccac acctccccct
     2761 gaacctgaaa cataaaatga atgcaattgt tgttgttaac ttgtttattg cagcttataa
     2821 tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca
     2881 ttctagttgt ggtttgtcca aactcatcaa tgtatcttaa ggcgtaaatt gtaagcgtta
     2941 atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg
     3001 ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg ttgagtgttg
     3061 ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa
     3121 aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca agttttttgg
     3181 ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt
     3241 gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg
     3301 ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta
     3361 atgcgccgct acagggcgcg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta
     3421 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
     3481 aaatgcttca ataatattga aaaaggaaga gtcctgaggc ggaaagaacc agctgtggaa
     3541 tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag
     3601 catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag
     3661 aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc
     3721 catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt
     3781 ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgagg
     3841 aggctttttt ggaggcctag gcttttgcaa agatcgatca agagacagga tgaggatcgt
     3901 ttcgcatgat tgaacaagat ggattgcacg caggttctcc ggccgcttgg gtggagaggc
     3961 tattcggcta tgactgggca caacagacaa tcggctgctc tgatgccgcc gtgttccggc
     4021 tgtcagcgca ggggcgcccg gttctttttg tcaagaccga cctgtccggt gccctgaatg
     4081 aactgcaaga cgaggcagcg cggctatcgt ggctggccac gacgggcgtt ccttgcgcag
     4141 ctgtgctcga cgttgtcact gaagcgggaa gggactggct gctattgggc gaagtgccgg
     4201 ggcaggatct cctgtcatct caccttgctc ctgccgagaa agtatccatc atggctgatg
     4261 caatgcggcg gctgcatacg cttgatccgg ctacctgccc attcgaccac caagcgaaac
     4321 atcgcatcga gcgagcacgt actcggatgg aagccggtct tgtcgatcag gatgatctgg
     4381 acgaagagca tcaggggctc gcgccagccg aactgttcgc caggctcaag gcgagcatgc
     4441 ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat atcatggtgg
     4501 aaaatggccg cttttctgga ttcatcgact gtggccggct gggtgtggcg gaccgctatc
     4561 aggacatagc gttggctacc cgtgatattg ctgaagagct tggcggcgaa tgggctgacc
     4621 gcttcctcgt gctttacggt atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc
     4681 ttcttgacga gttcttctga gcgggactct ggggttcgaa atgaccgacc aagcgacgcc
     4741 caacctgcca tcacgagatt tcgattccac cgccgccttc tatgaaaggt tgggcttcgg
     4801 aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt
     4861 cttcgcccac cctaggggga ggctaactga aacacggaag gagacaatac cggaaggaac
     4921 ccgcgctatg acggcaataa aaagacagaa taaaacgcac ggtgttgggt cgtttgttca
     4981 taaacgcggg gttcggtccc agggctggca ctctgtcgat accccaccga gaccccattg
     5041 gggccaatac gcccgcgttt cttccttttc cccaccccac cccccaagtt cgggtgaagg
     5101 cccagggctc gcagccaacg tcggggcggc aggccctgcc atagcctcag gttactcata
     5161 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct
     5221 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     5281 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     5341 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     5401 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct
     5461 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     5521 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     5581 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     5641 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     5701 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     5761 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     5821 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     5881 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     5941 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
     6001 cgccatgcat
//
