LOCUS       sequence-378567-        7767 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Stable expression of RvCAHS3 (fly codon optimized) in Drosophila 
            cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7767)
  AUTHORS   Tanaka A, Nakano T, Watanabe K, Masuda K, Honda G, Kamata S, Yasui 
            R, Kozuka-Hata H, Watanabe C, Chinen T, Kitagawa D, Sawai S, Oyama 
            M, Yanagisawa M, Kunieda T
  TITLE     Stress-dependent cell stiffening by tardigrade tolerance proteins 
            that reversibly form a filamentous network and gel.
  JOURNAL   PLoS Biol. 2022 Sep 6;20(9):e3001780. doi: 
            10.1371/journal.pbio.3001780. eCollection 2022 Sep.
  PUBMED    36067153
REFERENCE   2  (bases 1 to 7767)
  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: "PLoS 
            Biol."; date: "2022-09-6"; volume: "20(9):e3001780. doi"; pages: " 
            10.1371/journal.pbio.3001780. eCollection 2022 Sep"
COMMENT     Sequence Label: pAc5-STABLE2-neo-RvCAHS3
FEATURES             Location/Qualifiers
     source          1..7767
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      31..40
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             970..1023
                     /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"
     regulatory      1042..1051
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1048..1050,1051..1053,1054..1764)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKGD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1093..1114)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     1701..1722
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1768..1821
                     /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             1828..2631
                     /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="MGSAIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQ
                     GRPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQD
                     LLSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE
                     EHQGLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQ
                     DIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(1891..1910)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2501..2520
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    2639..2773
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2663..2682)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2717..2736
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      complement(3374..3962)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3454..3473)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4133..4924,4925..4993))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     4756..4775
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4994..5098)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5166..5184
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5248..7757
                     /label=Ac5 promoter
                     /note="Drosophila melanogaster actin 5C promoter"
ORIGIN
        1 ggtacctact agtccagtgt ggtggaattc gccaccatgg acatgagtag ccgccagaat
       61 cagcagagca gcagccagca tagtagcagc tcccagcaag gcggacaagg tggacagggt
      121 gttcaaggca gcagcagcta tagccgcacc gaggttcaca caagcagcgg cggaccaaca
      181 attggaggtg cccaacgtac agtgccagtg ccaccaggat cgcatagcga ggtgcacgaa
      241 gaacgcgaag tgatcaagca cggcaccaag accgagagcg agacacacgt tgtgaccgtg
      301 ccagtgacaa ccttcggcag caccaacatg gaaagcgtgc gcaccggatt caccgtgacg
      361 caggataaga acctgaccgt ggccgctccg aatatcgccg ctccaatcca cagcaacctg
      421 gatctgaatc tcggaggcgg agcccgtgcc gaaattaccg ctggaacaac ggtggatctg
      481 agcaagatcc agcgcaagga tctgggcccc gaagagtacg cccgttacaa ggccaaggtg
      541 gaacagctgg cccgccagga tgaacaggat gctggaatgc gtgccgcgca gtaccgtgaa
      601 gaggtggaac gtgatgccga gctgatccgc cagattctgg aacgccagca catccgcgat
      661 ttggagttcc gcaaagagat ggtcgagaat caagtgaacc gccaagagcg cgagatccag
      721 ttggaggccg aatatgccat gcgcgccctg gaactggaac gcaatgccgc caaagaagcc
      781 ctggaaagcg ccaaggctca gaccaacgtg aacgtgaagg tcgagagcgc catcggaacc
      841 acagtgtcca agggcgccat tcagaccagc gccgataaga gcagcaccac caagacagga
      901 cccaccaccg tgacacagat taagcacacc gagcagcata ccgagcgccg cctgtataaa
      961 ggcggccgcg aaggacgcgg cagcctactg acttgcggag atgtcgaaga gaaccctggc
     1021 cctggttccg atatctctag agccaccatg gtgagcaagg gcgaggagct gttcaccggg
     1081 gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc
     1141 ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc
     1201 ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc
     1261 ttcagccgct accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa
     1321 ggctacgtcc aggagcgcac catcttcttc aagggcgacg gcaactacaa gacccgcgcc
     1381 gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc
     1441 aaggaggacg gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc
     1501 tatatcatgg ccgacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac
     1561 atcgaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac
     1621 ggccccgtgc tgctgcccga caaccactac ctgagcaccc agtccgccct gagcaaagac
     1681 cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact
     1741 ctcggcatgg acgagctgta caagcttgag ggcagaggaa gtcttctaac atgcggtgac
     1801 gtggaggaga atcccggccc tgctagcatg ggatcggcca ttgaacaaga tggattgcac
     1861 gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca
     1921 atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt
     1981 gtcaagaccg acctgtccgg tgccctgaat gaactgcaag acgaggcagc gcggctatcg
     2041 tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga
     2101 agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct
     2161 cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg
     2221 gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg
     2281 gaagccggtc ttgtcgatca ggatgatctg gacgaagaac atcaggggct cgcgccagcc
     2341 gaactgttcg ccaggctcaa ggcgagcatg cccgacggcg aggatctcgt cgtgacccat
     2401 ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac
     2461 tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt
     2521 gctgaagaac ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct
     2581 cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg actcgaggga
     2641 tccagacatg ataagataca ttgatgagtt tggacaaacc acaactagaa tgcagtgaaa
     2701 aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta tttgtaacca ttataagctg
     2761 caataaacaa gttaacaaca acaattgcat tcattttatg tttcaggttc agggggaggt
     2821 gtgggaggtt ttttaaagca agtaaaacct ctacaaatgt ggtatggctg attatgatca
     2881 gtcgaccgat gcccttgaga gccttcaacc cagtcagctc cttccggtgg gcgcggggca
     2941 tgactatcgt cgccgcactt atgactgtct tctttatcat gcaactcgta ggacaggtgc
     3001 cggcagcgct ctgggtcatt ttcggcgagg accgctttcg ctggagcgcg acgatgatcg
     3061 gcctgtcgct tgcggtattc ggaatcttgc acgccctcgc tcaagccttc gtcactggtc
     3121 ccgccaccaa acgtttcggc gagaagcagg ccattatcgc cggcatggcg gccgacgcgc
     3181 tgggctacgt cttgctggcg ttcgcgacgc gaggctggat ggccttcccc attatgattc
     3241 ttctcgcttc cggcggcatc gggatgcccg cgttgcaggc catgctgtcc aggcaggtag
     3301 atgacgacca tcagggacag cttcaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     3361 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     3421 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     3481 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     3541 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     3601 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     3661 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     3721 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     3781 tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc
     3841 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     3901 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     3961 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     4021 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     4081 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     4141 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     4201 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     4261 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     4321 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     4381 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     4441 ccattgctgc aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     4501 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     4561 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     4621 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     4681 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     4741 cggcgtcaac acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     4801 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     4861 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     4921 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     4981 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     5041 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     5101 catttccccg aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct
     5161 ataaaaatag gcgtatcacg aggccctttc gtcttcaaga attaattcta tattctaaaa
     5221 acacaaatga tacttctaaa aaaaaatcat gaatggcatc aactctgaat caaatctttg
     5281 cagatgcacc tacttctcat ttccactgtc acatcatttt tccagatctc gctgcctgtt
     5341 atgtggccca caaaccaaga cacgttttat ggccattaaa gctggctgat cgtcgccaaa
     5401 caccaaatac ataatgaata tgtacacatt cgagaaagaa gcgatcaaag aagcgtcttc
     5461 gggcggagta ggagaatgcg gaggagaagg agaacgagct gatctagtat ctctccacaa
     5521 tccaatgcca actgaccaac tggccatatt cggagcaatt tgaagccaat ttccatcgcc
     5581 tggcgatcgc tccattcttg gctatatgtt tttcaccgtt acccggggcc attttcaaag
     5641 actcgtcggc aagataagat tgtgtcactc gctgtctctc ttcatttgtc gaagaatgct
     5701 gaggaatttc gcgatgacgt cggcgagtat tttgaagaat gagaataatt tgtatttata
     5761 cgaaaatcag ttagtggaat tttctacaaa aacatgttat ctatagataa ttttgttgca
     5821 aaatatgttg actatgacaa agattgtatg tatatacctt taatgtattc tcattttctt
     5881 atgtatttat aatggcaatg atgatactga tgatatttta agatgatgcc agaccaaaag
     5941 gcttgaattt ctgcgtcttt tgccgaacgc agtgcatgtg caattgttgt tttttggaat
     6001 attcaatttt cggactgtcc gctttgattt cagtttcttg gcttattcaa aaagcaaagt
     6061 aaagccaaaa aagcgagatg gcaataccaa atgcggcaaa acggtagtgg aaggaaaggg
     6121 gtgcggggca gcggaaggaa gggtggggcg gggcgtggcg gggtctgtgg ctgggcgcga
     6181 cgtcaccgac gttggagcca ctcctttgac catgtgtgcg tgtgtgtatt attcgtgtct
     6241 cgccactcgc cggttgtttt tttcttttta tgctgcgctc tctctagcgc catctcgctt
     6301 acgcatgctc aacgcaccgc atgttgccgt ttccttttat gcgtcatttt ggctcgaaat
     6361 aggcaattat ttaaacaaag attagtcaac gaaaacgcta aaataaataa gtctacaata
     6421 tggttactta ttgccatgtg tgtgcagcca acgatagcaa caaaagcaac aacacaggtg
     6481 gctttccctc tttcactttt tgtttgcaag ccgcgtgcga gcaagacggc acgaccggca
     6541 aacgcaatta cgctgacaaa gagcagacga agttttggcg aaaaacatca aggcgcctga
     6601 tacgaatgca tttgcaataa caattgcgat atttaatatt gtttatgaag ctgtttgact
     6661 tcaaaacaca caaaaaaaaa aataaaacaa attatttgaa agagaattag gaatcggacg
     6721 cttatcgtta gggtaacaac aagaaatgct tactgagtca cagcctctgg aaaactgccg
     6781 caagccagag agagagagaa aaagagggag agcagcttag accgcatgtg cttgtgtgtg
     6841 aggcgtctct ctcttcgtct ctgttgcgca aacgcataga ctgcactgaa aaaatcgatt
     6901 acctattttt tatgaatgaa tatttgcact attactattc aaaactatta agatagcaat
     6961 cacattcaat agccaaatac tataccacct gagcgatgca acgaaatgat caatttgagc
     7021 aaaaatgctg catatttagg acggcatcat tatagaaatg cttcttgctg tgtacttttc
     7081 tctcgtctgg cagctgtttc gccgttattg ttaaaaccgg cttaagttag gtgtgttttc
     7141 tacgactagt gaatgcccta ctagaagatg tgtgttgcac aaaatgtccc tggaataacc
     7201 aatttgaagt gcagatagca gtaaacgtaa gctaatatga atattattta actgtaatgt
     7261 tttaatatcg ctggacatta ctaataaacc cactataaac acatgtacat atgtatgttt
     7321 tggcatacaa tgagtagttg gggaaaaaat gtgtaaaagc accgtgacca tcacagcata
     7381 aagataacca gctgaagtat cgaatatgag taacccccaa attgaatcac atgccgcaac
     7441 tgataggacc catggaagta cactcttcat ggcgatatac aagacacaca caagcacgaa
     7501 cacccagttg cggaggaaat tctccgtaaa tgaaaaccca atcggcgaac aattcatacc
     7561 catatatggt aaaagttttg aacgcgactt gagagcggag agcattgcgg ctgataaggt
     7621 tttagcgcta agcgggcttt ataaaacggg ctgcgggacc agttttcata tcactaccgt
     7681 ttgagttctt gtgctgtgtg gatactcctc ccgacacaaa gccgctccat cagccagcag
     7741 tcgtctaatc cagagacccc ggatcgg
//
