LOCUS       sequence-378560-        6793 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of RvCAHS8.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6793)
  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 6793)
  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: pEThT-RvCAHS8
FEATURES             Location/Qualifiers
     source          1..6793
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             7..24
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             741..758
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             759..779
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             780..1433
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="SPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKF
                     ELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYG
                     VSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLY
                     MDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     CDS             1440..1457
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1510..1528)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1524..1571
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1608..2063
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1695..1714)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1905..1926
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2089..2193
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2194..2262,2263..3054)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2412..2431)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3225..3813
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3714..3733
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3967..3984
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3999..4138
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4064..4083)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4224..4246
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4240..4431)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5240..6322)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     6284..6303
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6323..6400)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6606..6625
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6709..6728
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6709..6727
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6728..6752
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     RBS             6767..6789
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 catatgcacc atcaccatca ccatggatca atgtctcagc gagcttcgga acactcatca
       61 tccagccacc agtcgtctgg ccagtctgga cagtcgagcc agcacgtcca agagcagaac
      121 accagctaca cacacaccga tgtccgcacc aacgttccca atgtccctcc tccagtcatc
      181 tcgaccagcg ctgctggtct cagtgagcag atggtcggtc aaggtttcac cgcctcggct
      241 gcgcgagtct ccggccagtc caccgaagcc catgtccaaa tgacccctga gatggaagcc
      301 gaggcccgca aggaccgcga acgatacgag cgcgaactgg aagccatcaa ccaacgccac
      361 cagaaggata ttgaaggcaa gaccgaggct taccgaaagc aagccgaagc tgaggcggaa
      421 cgtcttcgac gggaactaga gaagcaacat caacgagacg ttgagttccg caagtccctg
      481 gttcaaggga caatcgacaa ccagaagaaa caggttgagc tggaggctat gatggccaag
      541 cgcgagttgg accgagaagc caagctggct caagatgctc tcgagaagag caaaatggcc
      601 accgatgctc aagtcacgtt cgagtcagca gtaggccaca cgatttctgg tagcaccacc
      661 gtgtcccagt ccgagaaagt atcccagtcc aagaagtaaa attcgatatc aagcttatcg
      721 atgctagcac tagtcctagg ctggttccgc gtgggtccga gaacctgtac ttccagggct
      781 cccctatact aggttattgg aaaattaagg gccttgtgca acccactcga cttcttttgg
      841 aatatcttga agaaaaatat gaagagcatt tgtatgagcg cgatgaaggt gataaatggc
      901 gaaacaaaaa gtttgaattg ggtttggagt ttcccaatct tccttattat attgatggtg
      961 atgttaaatt aacacagtct atggccatca tacgttatat agctgacaag cacaacatgt
     1021 tgggtggttg tccaaaagag cgtgcagaga tttcaatgct tgaaggagcg gttttggata
     1081 ttagatacgg tgtttcgaga attgcatata gtaaagactt tgaaactctc aaagttgatt
     1141 ttcttagcaa gctacctgaa atgctgaaaa tgttcgaaga tcgtttatgt cataaaacat
     1201 atttaaatgg tgatcatgta acccatcctg acttcatgtt gtatgacgct cttgatgttg
     1261 ttttatacat ggacccaatg tgcctggatg cgttcccaaa attagtttgt tttaaaaaac
     1321 gtattgaagc tatcccacaa attgataagt acttgaaatc cagcaagtat atagcatggc
     1381 ctttgcaggg ctggcaagcc acgtttggtg gtggcgacca tcctccaaaa taactcgagc
     1441 accaccacca ccaccactga gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg
     1501 ctgctgccac cgctgagcaa taactagcat aaccccttgg ggcctctaaa cgggtcttga
     1561 ggggtttttt gctgaaagga ggaactatat ccggattggc gaatgggacg cgccctgtag
     1621 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
     1681 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
     1741 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     1801 cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata
     1861 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
     1921 aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc
     1981 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa
     2041 caaaatatta acgcttacaa tttaggtggc acttttcggg gaaatgtgcg cggaacccct
     2101 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
     2161 taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc
     2221 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg
     2281 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc
     2341 aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact
     2401 tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc
     2461 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag
     2521 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat
     2581 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt
     2641 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa
     2701 gccataccaa acgacgagcg tgacaccacg atgcctgcag caatggcaac aacgttgcgc
     2761 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg
     2821 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt
     2881 gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca
     2941 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat
     3001 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca
     3061 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
     3121 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     3181 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     3241 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     3301 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     3361 ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     3421 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     3481 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     3541 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     3601 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     3661 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     3721 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     3781 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     3841 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
     3901 gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc
     3961 gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt
     4021 acgcatctgt gcggtatttc acaccgcaat ggtgcactct cagtacaatc tgctctgatg
     4081 ccgcatagtt aagccagtat acactccgct atcgctacgt gactgggtca tggctgcgcc
     4141 ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc
     4201 ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc
     4261 accgaaacgc gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca
     4321 gatgtctgcc tgttcatccg cgtccagctc gttgagtttc tccagaagcg ttaatgtctg
     4381 gcttctgata aagcgggcca tgttaagggc ggttttttcc tgtttggtca ctgatgcctc
     4441 cgtgtaaggg ggatttctgt tcatgggggt aatgataccg atgaaacgag agaggatgct
     4501 cacgatacgg gttactgatg atgaacatgc ccggttactg gaacgttgtg agggtaaaca
     4561 actggcggta tggatgcggc gggaccagag aaaaatcact cagggtcaat gccagcgctt
     4621 cgttaataca gatgtaggtg ttccacaggg tagccagcag catcctgcga tgcagatccg
     4681 gaacataatg gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa cacggaaacc
     4741 gaagaccatt catgttgttg ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt
     4801 tcgctcgcgt atcggtgatt cattctgcta accagtaagg caaccccgcc agcctagccg
     4861 ggtcctcaac gacaggagca cgatcatgcg cacccgtggg gccgccatgc cggcgataat
     4921 ggcctgcttc tcgccgaaac gtttggtggc gggaccagtg acgaaggctt gagcgagggc
     4981 gtgcaagatt ccgaataccg caagcgacag gccgatcatc gtcgcgctcc agcgaaagcg
     5041 gtcctcgccg aaaatgaccc agagcgctgc cggcacctgt cctacgagtt gcatgataaa
     5101 gaagacagtc ataagtgcgg cgacgatagt catgccccgc gcccaccgga aggagctgac
     5161 tgggttgaag gctctcaagg gcatcggtcg agatcccggt gcctaatgag tgagctaact
     5221 tacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct
     5281 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gccagggtgg
     5341 tttttctttt caccagtgag acgggcaaca gctgattgcc cttcaccgcc tggccctgag
     5401 agagttgcag caagcggtcc acgctggttt gccccagcag gcgaaaatcc tgtttgatgg
     5461 tggttaacgg cgggatataa catgagctgt cttcggtatc gtcgtatccc actaccgaga
     5521 tatccgcacc aacgcgcagc ccggactcgg taatggcgcg cattgcgccc agcgccatct
     5581 gatcgttggc aaccagcatc gcagtgggaa cgatgccctc attcagcatt tgcatggttt
     5641 gttgaaaacc ggacatggca ctccagtcgc cttcccgttc cgctatcggc tgaatttgat
     5701 tgcgagtgag atatttatgc cagccagcca gacgcagacg cgccgagaca gaacttaatg
     5761 ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc acgcccagtc
     5821 gcgtaccgtc ttcatgggag aaaataatac tgttgatggg tgtctggtca gagacatcaa
     5881 gaaataacgc cggaacatta gtgcaggcag cttccacagc aatggcatcc tggtcatcca
     5941 gcggatagtt aatgatcagc ccactgacgc gttgcgcgag aagattgtgc accgccgctt
     6001 tacaggcttc gacgccgctt cgttctacca tcgacaccac cacgctggca cccagttgat
     6061 cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc agactggagg
     6121 tggcaacgcc aatcagcaac gactgtttgc ccgccagttg ttgtgccacg cggttgggaa
     6181 tgtaattcag ctccgccatc gccgcttcca ctttttcccg cgttttcgca gaaacgtggc
     6241 tggcctggtt caccacgcgg gaaacggtct gataagagac accggcatac tctgcgacat
     6301 cgtataacgt tactggtttc acattcacca ccctgaattg actctcttcc gggcgctatc
     6361 atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg acgctctccc
     6421 ttatgcgact cctgcattag gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc
     6481 gccgcaagga atggtgcatg caaggagatg gcgcccaaca gtcccccggc cacggggcct
     6541 gccaccatac ccacgccgaa acaagcgctc atgagcccga agtggcgagc ccgatcttcc
     6601 ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg
     6661 gccacgatgc gtccggcgta gaggatcgag atctcgatcc cgcgaaatta atacgactca
     6721 ctatagggga attgtgagcg gataacaatt cccctctaga aataattttg tttaacttta
     6781 agaaggagat ata
//
