LOCUS       sequence-378565-        6767 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Transient expression of RvCAHS8 (fly codon 
            optimized)-GS_linker-AcGFP1 in Drosophila cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6767)
  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 6767)
  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: pAc5.1/V5-HisA-RvCAHS8-GS-AcGFP1
FEATURES             Location/Qualifiers
     source          1..6767
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        56..2567
                     /label=Ac5 promoter
                     /note="Drosophila melanogaster actin 5C promoter"
     CDS             3324..4043
                     /codon_start=1
                     /product="Aequorea coerulescens GFP"
                     /label=AcGFP1
                     /note="mammalian codon-optimized"
                     /translation="MVSKGAELFTGIVPILIELNGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLSYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFEDD
                     GNYKSRAEVKFEGDTLVNRIELTGTDFKEDGNILGNKMEYNYNAHNVYIMTDKAKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMIYF
                     GFVTAAAITHGMDELYK"
     CDS             4073..4114
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             4124..4141
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4164..4181)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4214..4348
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4238..4257)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4292..4311
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      complement(4949..5537)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5029..5048)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5708..6499,6500..6568))
                     /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     6331..6350
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6569..6673)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6741..6759
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 cttcaagaat taattctata ttctaaaaac acaaatgata cttctaaaaa aaaatcatga
       61 atggcatcaa ctctgaatca aatctttgca gatgcaccta cttctcattt ccactgtcac
      121 atcatttttc cagatctcgc tgcctgttat gtggcccaca aaccaagaca cgttttatgg
      181 ccattaaagc tggctgatcg tcgccaaaca ccaaatacat aatgaatatg tacacattcg
      241 agaaagaagc gatcaaagaa gcgtcttcgg gcggagtagg agaatgcgga ggagaaggag
      301 aacgagctga tctagtatct ctccacaatc caatgccaac tgaccaactg gccatattcg
      361 gagcaatttg aagccaattt ccatcgcctg gcgatcgctc cattcttggc tatatgtttt
      421 tcaccgttac ccggggccat tttcaaagac tcgtcggcaa gataagattg tgtcactcgc
      481 tgtctctctt catttgtcga agaatgctga ggaatttcgc gatgacgtcg gcgagtattt
      541 tgaagaatga gaataatttg tatttatacg aaaatcagtt agtggaattt tctacaaaaa
      601 catgttatct atagataatt ttgttgcaaa atatgttgac tatgacaaag attgtatgta
      661 tataccttta atgtattctc attttcttat gtatttataa tggcaatgat gatactgatg
      721 atattttaag atgatgccag accaaaaggc ttgaatttct gcgtcttttg ccgaacgcag
      781 tgcatgtgca attgttgttt tttggaatat tcaattttcg gactgtccgc tttgatttca
      841 gtttcttggc ttattcaaaa agcaaagtaa agccaaaaaa gcgagatggc aataccaaat
      901 gcggcaaaac ggtagtggaa ggaaaggggt gcggggcagc ggaaggaagg gtggggcggg
      961 gcgtggcggg gtctgtggct gggcgcgacg tcaccgacgt tggagccact cctttgacca
     1021 tgtgtgcgtg tgtgtattat tcgtgtctcg ccactcgccg gttgtttttt tctttttatg
     1081 ctgcgctctc tctagcgcca tctcgcttac gcatgctcaa cgcaccgcat gttgccgttt
     1141 ccttttatgc gtcattttgg ctcgaaatag gcaattattt aaacaaagat tagtcaacga
     1201 aaacgctaaa ataaataagt ctacaatatg gttacttatt gccatgtgtg tgcagccaac
     1261 gatagcaaca aaagcaacaa cacaggtggc tttccctctt tcactttttg tttgcaagcc
     1321 gcgtgcgagc aagacggcac gaccggcaaa cgcaattacg ctgacaaaga gcagacgaag
     1381 ttttggcgaa aaacatcaag gcgcctgata cgaatgcatt tgcaataaca attgcgatat
     1441 ttaatattgt ttatgaagct gtttgacttc aaaacacaca aaaaaaaaaa aataaaacaa
     1501 attatttgaa agagaattag gaatcggacg cttatcgtta gggtaacaac aagaaatgct
     1561 tactgagtca cagcctctgg aaaactgccg caagccagag agagagagaa aaagagggag
     1621 agcagcttag accgcatgtg cttgtgtgtg aggcgtctct ctcttcgtct ctgttgcgca
     1681 aacgcataga ctgcactgaa aaaatcgatt acctattttt tatgaatgaa tatttgcact
     1741 attactattc aaaactatta agatagcaat cacattcaat agccaaatac tataccacct
     1801 gagcgatgca acgaaatgat caatttgagc aaaaatgctg catatttagg acggcatcat
     1861 tatagaaatg cttcttgctg tgtacttttc tctcgtctgg cagctgtttc gccgttattg
     1921 ttaaaaccgg cttaagttag gtgtgttttc tacgactagt gaatgcccta ctagaagatg
     1981 tgtgttgcac aaaatgtccc tggaataacc aatttgaagt gcagatagca gtaaacgtaa
     2041 gctaatatga atattattta actgtaatgt tttaatatcg ctggacatta ctaataaacc
     2101 cactataaac acatgtacat atgtatgttt tggcatacaa tgagtagttg gggaaaaaat
     2161 gtgtaaaagc accgtgacca tcacagcata aagataacca gctgaagtat cgaatatgag
     2221 taacccccaa attgaatcac atgccgcaac tgataggacc catggaagta cactcttcat
     2281 ggcgatatac aagacacaca caagcacgaa cacccagttg cggaggaaat tctccgtaaa
     2341 tgaaaaccca atcggcgaac aattcatacc catatatggt aaaagttttg aacgcgactt
     2401 gagagcggag agcattgcgg ctgataaggt tttagcgcta agcgggcttt ataaaacggg
     2461 ctgcgggacc agttttcata tcactaccgt ttgagttctt gtgctgtgtg gatactcctc
     2521 ccgacacaaa gccgctccat cagccagcag tcgtctaatc cagagacccc ggatcggggt
     2581 acctactagt ccagtgtggt ggaattgcca ccatgagtca gcgcgccagc gaacatagca
     2641 gcagcagtca tcagagcagc ggccagagtg gacagagcag ccagcatgtg caagagcaga
     2701 acaccagcta cacccacacc gatgtgcgca ccaacgtgcc caatgtgcca ccaccagtga
     2761 tcagtacaag tgccgccgga ctgagcgagc agatggttgg acaaggattc acagccagcg
     2821 ccgctcgtgt tagcggacaa agtactgagg cccacgtgca gatgacccca gagatggaag
     2881 ccgaggctcg caaggatcgc gaacgctatg aacgcgagct ggaagccatc aaccagcgcc
     2941 accagaagga tatcgagggc aagacagagg cctaccgcaa gcaagccgaa gctgaagccg
     3001 aacgcctgcg ccgtgaactg gaaaagcagc accagcgcga cgtcgagttt cgcaagagtc
     3061 tggtgcaggg caccatcgac aaccagaaga agcaggtcga gttggaggcc atgatggcca
     3121 agcgcgaact ggatcgtgaa gccaagctgg cccaagatgc cctggaaaag tccaagatgg
     3181 ccacggatgc ccaagtgacc tttgagagcg ccgtgggcca taccattagc ggcagtacaa
     3241 cagtgtccca gagcgagaag gtgtcccagt ccaagaaggg tggaggcggt tcaggcggag
     3301 gtggctctgg cggtggcgga tcgatggtga gcaagggcgc cgagctgttc accggcatcg
     3361 tgcccatcct gatcgagctg aatggcgatg tgaatggcca caagttcagc gtgagcggcg
     3421 agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca
     3481 agctgcctgt gccctggccc accctggtga ccaccctgag ctacggcgtg cagtgcttct
     3541 cacgctaccc cgatcacatg aagcagcacg acttcttcaa gagcgccatg cctgagggct
     3601 acatccagga gcgcaccatc ttcttcgagg atgacggcaa ctacaagtcg cgcgccgagg
     3661 tgaagttcga gggcgatacc ctggtgaatc gcatcgagct gaccggcacc gatttcaagg
     3721 aggatggcaa catcctgggc aataagatgg agtacaacta caacgcccac aatgtgtaca
     3781 tcatgaccga caaggccaag aatggcatca aggtgaactt caagatccgc cacaacatcg
     3841 aggatggcag cgtgcagctg gccgaccact accagcagaa tacccccatc ggcgatggcc
     3901 ctgtgctgct gcccgataac cactacctgt ccacccagag cgccctgtcc aaggacccca
     3961 acgagaagcg cgatcacatg atctacttcg gcttcgtgac cgccgccgcc atcacccacg
     4021 gcatggatga gctgtacaag tgaggccgct cgagtctaga gggcccttcg aaggtaagcc
     4081 tatccctaac cctctcctcg gtctcgattc tacgcgtacc ggtcatcatc accatcacca
     4141 ttgagtttaa acccgctgat cagcctcgac tgtgccttct aaggcctgag ctcgctgatc
     4201 agcctcgatc gaggatccag acatgataag atacattgat gagtttggac aaaccacaac
     4261 tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt gatgctattg ctttatttgt
     4321 aaccattata agctgcaata aacaagttaa caacaacaat tgcattcatt ttatgtttca
     4381 ggttcagggg gaggtgtggg aggtttttta aagcaagtaa aacctctaca aatgtggtat
     4441 ggctgattat gatcagtcga ccgatgccct tgagagcctt caacccagtc agctccttcc
     4501 ggtgggcgcg gggcatgact atcgtcgccg cacttatgac tgtcttcttt atcatgcaac
     4561 tcgtaggaca ggtgccggca gcgctctggg tcattttcgg cgaggaccgc tttcgctgga
     4621 gcgcgacgat gatcggcctg tcgcttgcgg tattcggaat cttgcacgcc ctcgctcaag
     4681 ccttcgtcac tggtcccgcc accaaacgtt tcggcgagaa gcaggccatt atcgccggca
     4741 tggcggccga cgcgctgggc tacgtcttgc tggcgttcgc gacgcgaggc tggatggcct
     4801 tccccattat gattcttctc gcttccggcg gcatcgggat gcccgcgttg caggccatgc
     4861 tgtccaggca ggtagatgac gaccatcagg gacagcttca aggccagcaa aaggccagga
     4921 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     4981 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     5041 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     5101 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     5161 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     5221 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     5281 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     5341 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg
     5401 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     5461 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     5521 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     5581 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     5641 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     5701 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     5761 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     5821 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     5881 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     5941 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     6001 tgcgcaacgt tgttgccatt gctgcaggca tcgtggtgtc acgctcgtcg tttggtatgg
     6061 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     6121 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     6181 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     6241 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     6301 cgagttgctc ttgcccggcg tcaacacggg ataataccgc gccacatagc agaactttaa
     6361 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     6421 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     6481 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     6541 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     6601 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     6661 taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtctaagaa accattatta
     6721 tcatgacatt aacctataaa aataggcgta tcacgaggcc ctttcgt
//
