LOCUS       sequence-378557-        5223 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of RvCAHS3-min-GS_linker-AcGFP1 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5223)
  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 5223)
  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-RvCAHS3-min-GS
FEATURES             Location/Qualifiers
     source          1..5223
                     /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             1170..1889
                     /codon_start=1
                     /product="Aequorea coerulescens GFP"
                     /label=AcGFP1
                     /note="mammalian codon-optimized"
                     /translation="MVSKGAELFTGIVPILIELNGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLSYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFEDD
                     GNYKSRAEVKFEGDTLVNRIELTGTDFKEDGNILGNKMEYNYNAHNVYIMTDKAKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMIYF
                     GFVTAAAITHGMDELYK"
     polyA_signal    2011..2132
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2048..2067)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2102..2121
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2139..2594)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2276..2297)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2488..2507
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2621..2725
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2727..3084
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2727..2918
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2750..2770)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2935..3070
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3119..3913
                     /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             3119..3913
                     /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(3173..3192)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3783..3802
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4101..4120)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4145..4192
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4521..5109
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5010..5029
                     /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 gccaccatgc
      601 gaaccggctt caccgtcacc caagacaaga acttgaccgt tgctgctccc aacatcgctg
      661 ctcccatcca cagcaacctc gaccttaacc tcggcggtgg agctcgcgct gaaattaccg
      721 cagggaccac cgttgacttg agcaagatcc agcgcaagga tttgggacct gaagagtatg
      781 ctcgctacaa ggccaaggtc gagcaactgg ccaggcaaga tgagcaagac gcgggtatgc
      841 gcgctgccca gtaccgagag gaagtggagc gtgatgccga actcatccga cagatcttgg
      901 agcgacaaca catccgtgat cttgaattcc gcaaggaaat ggttgagaac caagtcaacc
      961 gacaagagag agaaatccag ctggaggctg agtacgcaat gcgagccctc gagcttgagc
     1021 gcaatgccgc taaagaggct ttggaaagcg ccaaggccca gactaacgtc aatgtcaagg
     1081 tcgagtccgc tattggtacc accgtctcca agggtgcaat ccagggtgga ggcggttcag
     1141 gcggaggtgg ctctggcggt ggcggatcga tggtgagcaa gggcgccgag ctgttcaccg
     1201 gcatcgtgcc catcctgatc gagctgaatg gcgatgtgaa tggccacaag ttcagcgtga
     1261 gcggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca
     1321 ccggcaagct gcctgtgccc tggcccaccc tggtgaccac cctgagctac ggcgtgcagt
     1381 gcttctcacg ctaccccgat cacatgaagc agcacgactt cttcaagagc gccatgcctg
     1441 agggctacat ccaggagcgc accatcttct tcgaggatga cggcaactac aagtcgcgcg
     1501 ccgaggtgaa gttcgagggc gataccctgg tgaatcgcat cgagctgacc ggcaccgatt
     1561 tcaaggagga tggcaacatc ctgggcaata agatggagta caactacaac gcccacaatg
     1621 tgtacatcat gaccgacaag gccaagaatg gcatcaaggt gaacttcaag atccgccaca
     1681 acatcgagga tggcagcgtg cagctggccg accactacca gcagaatacc cccatcggcg
     1741 atggccctgt gctgctgccc gataaccact acctgtccac ccagagcgcc ctgtccaagg
     1801 accccaacga gaagcgcgat cacatgatct acttcggctt cgtgaccgcc gccgccatca
     1861 cccacggcat ggatgagctg tacaagtgag cggccgcgac tctagatcat aatcagccat
     1921 accacatttg tagaggtttt acttgcttta aaaaacctcc cacacctccc cctgaacctg
     1981 aaacataaaa tgaatgcaat tgttgttgtt aacttgttta ttgcagctta taatggttac
     2041 aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt
     2101 tgtggtttgt ccaaactcat caatgtatct taaggcgtaa attgtaagcg ttaatatttt
     2161 gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat
     2221 cggcaaaatc ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt
     2281 ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt
     2341 ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt tggggtcgag
     2401 gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg
     2461 aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc
     2521 gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc
     2581 gctacagggc gcgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
     2641 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct
     2701 tcaataatat tgaaaaagga agagtcctga ggcggaaaga accagctgtg gaatgtgtgt
     2761 cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat
     2821 ctcaattagt cagcaaccag gtgtggaaag tccccaggct ccccagcagg cagaagtatg
     2881 caaagcatgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg
     2941 cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt
     3001 tatgcagagg ccgaggccgc ctcggcctct gagctattcc agaagtagtg aggaggcttt
     3061 tttggaggcc taggcttttg caaagatcga tcaagagaca ggatgaggat cgtttcgcat
     3121 gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg
     3181 ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc
     3241 gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca
     3301 agacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct
     3361 cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga
     3421 tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg
     3481 gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat
     3541 cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga
     3601 gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgagca tgcccgacgg
     3661 cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg
     3721 ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat
     3781 agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct
     3841 cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga
     3901 cgagttcttc tgagcgggac tctggggttc gaaatgaccg accaagcgac gcccaacctg
     3961 ccatcacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt
     4021 ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc
     4081 caccctaggg ggaggctaac tgaaacacgg aaggagacaa taccggaagg aacccgcgct
     4141 atgacggcaa taaaaagaca gaataaaacg cacggtgttg ggtcgtttgt tcataaacgc
     4201 ggggttcggt cccagggctg gcactctgtc gataccccac cgagacccca ttggggccaa
     4261 tacgcccgcg tttcttcctt ttccccaccc caccccccaa gttcgggtga aggcccaggg
     4321 ctcgcagcca acgtcggggc ggcaggccct gccatagcct caggttactc atatatactt
     4381 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat
     4441 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     4501 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     4561 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     4621 tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag
     4681 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     4741 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     4801 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     4861 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     4921 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     4981 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     5041 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     5101 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     5161 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccatg
     5221 cat
//
