LOCUS       sequence-378553-        5604 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of RvCAHS3-GS_linker-AcGFP1 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5604)
  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 5604)
  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-GS
FEATURES             Location/Qualifiers
     source          1..5604
                     /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             1551..2270
                     /codon_start=1
                     /product="Aequorea coerulescens GFP"
                     /label=AcGFP1
                     /note="mammalian codon-optimized"
                     /translation="MVSKGAELFTGIVPILIELNGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLSYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFEDD
                     GNYKSRAEVKFEGDTLVNRIELTGTDFKEDGNILGNKMEYNYNAHNVYIMTDKAKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMIYF
                     GFVTAAAITHGMDELYK"
     polyA_signal    2392..2513
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2429..2448)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2483..2502
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2520..2975)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2657..2678)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2869..2888
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3002..3106
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3108..3465
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3108..3299
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3131..3151)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3316..3451
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3500..4294
                     /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             3500..4294
                     /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(3554..3573)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4164..4183
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4482..4501)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4526..4573
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4902..5490
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5391..5410
                     /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 cttcccgaca gaaccagcaa tcgtccagcc aacactcgtc ctccagccag caaggtggtc
      661 aaggtggtca aggtgttcaa ggaagttcca gctactcgcg caccgaggtc cacaccagca
      721 gtggaggacc taccatcggt ggagcccagc gaactgtccc cgtcccccct ggatctcact
      781 ccgaggtcca tgaggagcgt gaggtcatca agcatggtac caaaaccgaa agcgagaccc
      841 acgtcgtcac cgtcccagtg acaactttcg gcagcaccaa catggaatct gtccgaaccg
      901 gcttcaccgt cacccaagac aagaacttga ccgttgctgc tcccaacatc gctgctccca
      961 tccacagcaa cctcgacctt aacctcggcg gtggagctcg cgctgaaatt accgcaggga
     1021 ccaccgttga cttgagcaag atccagcgca aggatttggg acctgaagag tatgctcgct
     1081 acaaggccaa ggtcgagcaa ctggccaggc aagatgagca agacgcgggt atgcgcgctg
     1141 cccagtaccg agaggaagtg gagcgtgatg ccgaactcat ccgacagatc ttggagcgac
     1201 aacacatccg tgatcttgaa ttccgcaagg aaatggttga gaaccaagtc aaccgacaag
     1261 agagagaaat ccagctggag gctgagtacg caatgcgagc cctcgagctt gagcgcaatg
     1321 ccgctaaaga ggctttggaa agcgccaagg cccagactaa cgtcaatgtc aaggtcgagt
     1381 ccgctattgg taccaccgtc tccaagggtg caatccagac ttccgccgac aagagcagca
     1441 ccaccaagac tggacccacc accgtcactc agattaaaca taccgaacaa cacactgaac
     1501 gccgaggtgg aggcggttca ggcggaggtg gctctggcgg tggcggatcg atggtgagca
     1561 agggcgccga gctgttcacc ggcatcgtgc ccatcctgat cgagctgaat ggcgatgtga
     1621 atggccacaa gttcagcgtg agcggcgagg gcgagggcga tgccacctac ggcaagctga
     1681 ccctgaagtt catctgcacc accggcaagc tgcctgtgcc ctggcccacc ctggtgacca
     1741 ccctgagcta cggcgtgcag tgcttctcac gctaccccga tcacatgaag cagcacgact
     1801 tcttcaagag cgccatgcct gagggctaca tccaggagcg caccatcttc ttcgaggatg
     1861 acggcaacta caagtcgcgc gccgaggtga agttcgaggg cgataccctg gtgaatcgca
     1921 tcgagctgac cggcaccgat ttcaaggagg atggcaacat cctgggcaat aagatggagt
     1981 acaactacaa cgcccacaat gtgtacatca tgaccgacaa ggccaagaat ggcatcaagg
     2041 tgaacttcaa gatccgccac aacatcgagg atggcagcgt gcagctggcc gaccactacc
     2101 agcagaatac ccccatcggc gatggccctg tgctgctgcc cgataaccac tacctgtcca
     2161 cccagagcgc cctgtccaag gaccccaacg agaagcgcga tcacatgatc tacttcggct
     2221 tcgtgaccgc cgccgccatc acccacggca tggatgagct gtacaagtga gcggccgcga
     2281 ctctagatca taatcagcca taccacattt gtagaggttt tacttgcttt aaaaaacctc
     2341 ccacacctcc ccctgaacct gaaacataaa atgaatgcaa ttgttgttgt taacttgttt
     2401 attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca
     2461 tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttaaggcgta
     2521 aattgtaagc gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt
     2581 ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat
     2641 agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa
     2701 cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccta
     2761 atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc
     2821 ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc
     2881 gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac
     2941 acccgccgcg cttaatgcgc cgctacaggg cgcgtcaggt ggcacttttc ggggaaatgt
     3001 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
     3061 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtcctg aggcggaaag
     3121 aaccagctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc
     3181 agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc
     3241 tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg
     3301 cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat
     3361 ggctgactaa ttttttttat ttatgcagag gccgaggccg cctcggcctc tgagctattc
     3421 cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaagatcg atcaagagac
     3481 aggatgagga tcgtttcgca tgattgaaca agatggattg cacgcaggtt ctccggccgc
     3541 ttgggtggag aggctattcg gctatgactg ggcacaacag acaatcggct gctctgatgc
     3601 cgccgtgttc cggctgtcag cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc
     3661 cggtgccctg aatgaactgc aagacgaggc agcgcggcta tcgtggctgg ccacgacggg
     3721 cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg ggaagggact ggctgctatt
     3781 gggcgaagtg ccggggcagg atctcctgtc atctcacctt gctcctgccg agaaagtatc
     3841 catcatggct gatgcaatgc ggcggctgca tacgcttgat ccggctacct gcccattcga
     3901 ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg atggaagccg gtcttgtcga
     3961 tcaggatgat ctggacgaag agcatcaggg gctcgcgcca gccgaactgt tcgccaggct
     4021 caaggcgagc atgcccgacg gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc
     4081 gaatatcatg gtggaaaatg gccgcttttc tggattcatc gactgtggcc ggctgggtgt
     4141 ggcggaccgc tatcaggaca tagcgttggc tacccgtgat attgctgaag agcttggcgg
     4201 cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat
     4261 cgccttctat cgccttcttg acgagttctt ctgagcggga ctctggggtt cgaaatgacc
     4321 gaccaagcga cgcccaacct gccatcacga gatttcgatt ccaccgccgc cttctatgaa
     4381 aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat
     4441 ctcatgctgg agttcttcgc ccaccctagg gggaggctaa ctgaaacacg gaaggagaca
     4501 ataccggaag gaacccgcgc tatgacggca ataaaaagac agaataaaac gcacggtgtt
     4561 gggtcgtttg ttcataaacg cggggttcgg tcccagggct ggcactctgt cgatacccca
     4621 ccgagacccc attggggcca atacgcccgc gtttcttcct tttccccacc ccacccccca
     4681 agttcgggtg aaggcccagg gctcgcagcc aacgtcgggg cggcaggccc tgccatagcc
     4741 tcaggttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
     4801 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     4861 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     4921 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     4981 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     5041 aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     5101 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     5161 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     5221 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     5281 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     5341 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     5401 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     5461 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     5521 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
     5581 gataaccgta ttaccgccat gcat
//
