LOCUS       sequence-378556-        5586 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of RvCAHS12-GS_linker-AcGFP1 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5586)
  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 5586)
  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-RvCAHS12-GS
FEATURES             Location/Qualifiers
     source          1..5586
                     /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             1533..2252
                     /codon_start=1
                     /product="Aequorea coerulescens GFP"
                     /label=AcGFP1
                     /note="mammalian codon-optimized"
                     /translation="MVSKGAELFTGIVPILIELNGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLSYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFEDD
                     GNYKSRAEVKFEGDTLVNRIELTGTDFKEDGNILGNKMEYNYNAHNVYIMTDKAKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMIYF
                     GFVTAAAITHGMDELYK"
     polyA_signal    2374..2495
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2411..2430)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2465..2484
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2502..2957)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2639..2660)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2851..2870
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2984..3088
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3090..3447
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3090..3281
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3113..3133)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3298..3433
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3482..4276
                     /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             3482..4276
                     /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(3536..3555)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4146..4165
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4464..4483)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4508..4555
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4884..5472
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5373..5392
                     /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 ctcataccca cgaacaaaaa ttcgagcgcg tcgaggaacg caaggtggac gacaagaaag
      661 gtctgcagga agttcgcgtt ggagtcgata ctggtcacgg agatcccgct ttgaacttca
      721 ctcccacgga tgccaccttg gtgcgcaccg gtggcgtcgg tggtaccact gcaagcagcc
      781 attcctccca catgtcctct tcaaccggcg gagctgtcac cggagcttcg cagtactctt
      841 caaccatgca ccaggaagga ggacacatga cgacggaggc ctcgaagaac acttcgtaca
      901 ctcacaccga ggtccgagct ccagtcatcg ataccgcgcc tccgatcatc tccaccggag
      961 catctggcat ggccgaacag atcgtcggtc aaggtttcac ggcctctgca gctcgtatca
     1021 ctggctcgtc ggctgatgtc aacatcgttg agactgctga ggctcgcgag aagcgaatga
     1081 gggatgagga gaaatatgct cgtgaacagg aggccatcaa ccgtcatgct gacaaggacc
     1141 tggaaaagaa gacggaagcc taccgcaagg aggccgagca cgaggctgag aagatccgca
     1201 aggcgttgga gaagcaacac gagcgagaca tcgagttccg caaggaagtt gtgggaagta
     1261 ccatcgagaa gcagaaggcg gaaatcgagc tggaagccaa gcgagcaaag gccgctttgg
     1321 agcacgaacg tcagctggcc aacgatgcgt tggagcgttc taagatgcat accgatgtcc
     1381 aagtcacgat ggacactgca gccggacata ccgtcagcgg tggaaccacg atgtcgtcat
     1441 cggagcagca cagttcttcg cacagctcat ctcgcactgg cggactgggt ggaggcggtt
     1501 caggcggagg tggctctggc ggtggcggat cgatggtgag caagggcgcc gagctgttca
     1561 ccggcatcgt gcccatcctg atcgagctga atggcgatgt gaatggccac aagttcagcg
     1621 tgagcggcga gggcgagggc gatgccacct acggcaagct gaccctgaag ttcatctgca
     1681 ccaccggcaa gctgcctgtg ccctggccca ccctggtgac caccctgagc tacggcgtgc
     1741 agtgcttctc acgctacccc gatcacatga agcagcacga cttcttcaag agcgccatgc
     1801 ctgagggcta catccaggag cgcaccatct tcttcgagga tgacggcaac tacaagtcgc
     1861 gcgccgaggt gaagttcgag ggcgataccc tggtgaatcg catcgagctg accggcaccg
     1921 atttcaagga ggatggcaac atcctgggca ataagatgga gtacaactac aacgcccaca
     1981 atgtgtacat catgaccgac aaggccaaga atggcatcaa ggtgaacttc aagatccgcc
     2041 acaacatcga ggatggcagc gtgcagctgg ccgaccacta ccagcagaat acccccatcg
     2101 gcgatggccc tgtgctgctg cccgataacc actacctgtc cacccagagc gccctgtcca
     2161 aggaccccaa cgagaagcgc gatcacatga tctacttcgg cttcgtgacc gccgccgcca
     2221 tcacccacgg catggatgag ctgtacaagt gagcggccgc gactctagat cataatcagc
     2281 cataccacat ttgtagaggt tttacttgct ttaaaaaacc tcccacacct ccccctgaac
     2341 ctgaaacata aaatgaatgc aattgttgtt gttaacttgt ttattgcagc ttataatggt
     2401 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct
     2461 agttgtggtt tgtccaaact catcaatgta tcttaaggcg taaattgtaa gcgttaatat
     2521 tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga
     2581 aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc
     2641 agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac
     2701 cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt ttttggggtc
     2761 gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg
     2821 gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag
     2881 ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc
     2941 gccgctacag ggcgcgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     3001 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
     3061 gcttcaataa tattgaaaaa ggaagagtcc tgaggcggaa agaaccagct gtggaatgtg
     3121 tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg
     3181 catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt
     3241 atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc
     3301 ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt
     3361 atttatgcag aggccgaggc cgcctcggcc tctgagctat tccagaagta gtgaggaggc
     3421 ttttttggag gcctaggctt ttgcaaagat cgatcaagag acaggatgag gatcgtttcg
     3481 catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt
     3541 cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc
     3601 agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact
     3661 gcaagacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt
     3721 gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca
     3781 ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat
     3841 gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg
     3901 catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga
     3961 agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcga gcatgcccga
     4021 cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa
     4081 tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga
     4141 catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt
     4201 cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct
     4261 tgacgagttc ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc gacgcccaac
     4321 ctgccatcac gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc
     4381 gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc
     4441 gcccacccta gggggaggct aactgaaaca cggaaggaga caataccgga aggaacccgc
     4501 gctatgacgg caataaaaag acagaataaa acgcacggtg ttgggtcgtt tgttcataaa
     4561 cgcggggttc ggtcccaggg ctggcactct gtcgataccc caccgagacc ccattggggc
     4621 caatacgccc gcgtttcttc cttttcccca ccccaccccc caagttcggg tgaaggccca
     4681 gggctcgcag ccaacgtcgg ggcggcaggc cctgccatag cctcaggtta ctcatatata
     4741 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
     4801 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     4861 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     4921 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     4981 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg
     5041 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     5101 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     5161 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     5221 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     5281 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     5341 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     5401 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     5461 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     5521 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
     5581 atgcat
//
