LOCUS       sequence-378554-        5360 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of RvCAHS8-GS_linker-AcGFP1 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5360)
  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 5360)
  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: pAcGFP1-N1-RvCAHS8-GS
FEATURES             Location/Qualifiers
     source          1..5360
                     /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"
     CDS             1308..2027
                     /codon_start=1
                     /product="Aequorea coerulescens GFP"
                     /label=AcGFP1
                     /note="mammalian codon-optimized"
                     /translation="MVSKGAELFTGIVPILIELNGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLSYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFEDD
                     GNYKSRAEVKFEGDTLVNRIELTGTDFKEDGNILGNKMEYNYNAHNVYIMTDKAKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMIYF
                     GFVTAAAITHGMDELYK"
     polyA_signal    2149..2270
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2186..2205)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2240..2259
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2277..2732)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2414..2435)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2626..2645
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2759..2863
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2865..3222
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2865..3056
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2888..2908)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3073..3208
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3257..4051
                     /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             3257..4051
                     /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(3311..3330)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3921..3940
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4239..4258)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4283..4330
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4659..5246
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5147..5166
                     /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 gccaccatgt
      601 ctcagcgagc ttcggaacac tcatcatcca gccaccagtc gtctggccag tctggacagt
      661 cgagccagca cgtccaagag cagaacacca gctacacaca caccgatgtc cgcaccaacg
      721 ttcccaatgt ccctcctcca gtcatctcga ccagcgctgc tggtctcagt gagcagatgg
      781 tcggtcaagg tttcaccgcc tcggctgcgc gagtctccgg ccagtccacc gaagcccatg
      841 tccaaatgac ccctgagatg gaagccgagg cccgcaagga ccgcgaacga tacgagcgcg
      901 aactggaagc catcaaccaa cgccaccaga aggatattga aggcaagacc gaggcttacc
      961 gaaagcaagc cgaagctgag gcggaacgtc ttcgacggga actagagaag caacatcaac
     1021 gagacgttga gttccgcaag tccctggttc aagggacaat cgacaaccag aagaaacagg
     1081 ttgagctgga ggctatgatg gccaagcgcg agttggaccg agaagccaag ctggctcaag
     1141 atgctctcga gaagagcaaa atggccaccg atgctcaagt cacgttcgag tcagcagtag
     1201 gccacacgat ttctggtagc accaccgtgt cccagtccga gaaagtatcc cagtccaaga
     1261 agggtggagg cggttcaggc ggaggtggct ctggcggtgg cggatcgatg gtgagcaagg
     1321 gcgccgagct gttcaccggc atcgtgccca tcctgatcga gctgaatggc gatgtgaatg
     1381 gccacaagtt cagcgtgagc ggcgagggcg agggcgatgc cacctacggc aagctgaccc
     1441 tgaagttcat ctgcaccacc ggcaagctgc ctgtgccctg gcccaccctg gtgaccaccc
     1501 tgagctacgg cgtgcagtgc ttctcacgct accccgatca catgaagcag cacgacttct
     1561 tcaagagcgc catgcctgag ggctacatcc aggagcgcac catcttcttc gaggatgacg
     1621 gcaactacaa gtcgcgcgcc gaggtgaagt tcgagggcga taccctggtg aatcgcatcg
     1681 agctgaccgg caccgatttc aaggaggatg gcaacatcct gggcaataag atggagtaca
     1741 actacaacgc ccacaatgtg tacatcatga ccgacaaggc caagaatggc atcaaggtga
     1801 acttcaagat ccgccacaac atcgaggatg gcagcgtgca gctggccgac cactaccagc
     1861 agaatacccc catcggcgat ggccctgtgc tgctgcccga taaccactac ctgtccaccc
     1921 agagcgccct gtccaaggac cccaacgaga agcgcgatca catgatctac ttcggcttcg
     1981 tgaccgccgc cgccatcacc cacggcatgg atgagctgta caagtgagcg gccgcgactc
     2041 tagatcataa tcagccatac cacatttgta gaggttttac ttgctttaaa aaacctccca
     2101 cacctccccc tgaacctgaa acataaaatg aatgcaattg ttgttgttaa cttgtttatt
     2161 gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt
     2221 ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta aggcgtaaat
     2281 tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt
     2341 taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg
     2401 gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt
     2461 caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc
     2521 aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg
     2581 atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa
     2641 aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc
     2701 cgccgcgctt aatgcgccgc tacagggcgc gtcaggtggc acttttcggg gaaatgtgcg
     2761 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
     2821 ataaccctga taaatgcttc aataatattg aaaaaggaag agtcctgagg cggaaagaac
     2881 cagctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc agcaggcaga
     2941 agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc cccaggctcc
     3001 ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgccc
     3061 ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc
     3121 tgactaattt tttttattta tgcagaggcc gaggccgcct cggcctctga gctattccag
     3181 aagtagtgag gaggcttttt tggaggccta ggcttttgca aagatcgatc aagagacagg
     3241 atgaggatcg tttcgcatga ttgaacaaga tggattgcac gcaggttctc cggccgcttg
     3301 ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc
     3361 cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg
     3421 tgccctgaat gaactgcaag acgaggcagc gcggctatcg tggctggcca cgacgggcgt
     3481 tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg
     3541 cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat
     3601 catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca
     3661 ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca
     3721 ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa
     3781 ggcgagcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa
     3841 tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc
     3901 ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga
     3961 atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc
     4021 cttctatcgc cttcttgacg agttcttctg agcgggactc tggggttcga aatgaccgac
     4081 caagcgacgc ccaacctgcc atcacgagat ttcgattcca ccgccgcctt ctatgaaagg
     4141 ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc
     4201 atgctggagt tcttcgccca ccctaggggg aggctaactg aaacacggaa ggagacaata
     4261 ccggaaggaa cccgcgctat gacggcaata aaaagacaga ataaaacgca cggtgttggg
     4321 tcgtttgttc ataaacgcgg ggttcggtcc cagggctggc actctgtcga taccccaccg
     4381 agaccccatt ggggccaata cgcccgcgtt tcttcctttt ccccacccca ccccccaagt
     4441 tcgggtgaag gcccagggct cgcagccaac gtcggggcgg caggccctgc catagcctca
     4501 ggttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
     4561 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     4621 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     4681 gtaatctgct gcttgcaaac aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     4741 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     4801 ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     4861 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     4921 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     4981 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     5041 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     5101 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     5161 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     5221 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     5281 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     5341 accgtattac cgccatgcat
//
