LOCUS       sequence-378561-        7018 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of RvCAHS12.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7018)
  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 7018)
  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-RvCAHS12
FEATURES             Location/Qualifiers
     source          1..7018
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             7..24
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             966..983
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             984..1004
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1005..1658
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="SPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKF
                     ELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYG
                     VSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLY
                     MDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     CDS             1665..1682
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1735..1753)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1749..1796
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1833..2288
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1920..1939)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2130..2151
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2314..2418
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2419..2487,2488..3279)
                     /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(2637..2656)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3450..4038
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3939..3958
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4192..4209
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4224..4363
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4289..4308)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4449..4471
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4465..4656)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5465..6547)
                     /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     6509..6528
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6548..6625)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6831..6850
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6934..6953
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6934..6952
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6953..6977
                     /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             6992..7014
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 catatgcacc atcaccatca ccatggatca atgtctcata cccacgaaca aaaattcgag
       61 cgcgtcgagg aacgcaaggt ggacgacaag aaaggtctgc aggaagttcg cgttggagtc
      121 gatactggtc acggagatcc cgctttgaac ttcactccca cggatgccac cttggtgcgc
      181 accggtggcg tcggtggtac cactgcaagc agccattcct cccacatgtc ctcttcaacc
      241 ggcggagctg tcaccggagc ttcgcagtac tcttcaacca tgcaccagga aggaggacac
      301 atgacgacgg aggcctcgaa gaacacttcg tacactcaca ccgaggtccg agctccagtc
      361 atcgataccg cgcctccgat catctccacc ggagcatctg gcatggccga acagatcgtc
      421 ggtcaaggtt tcacggcctc tgcagctcgt atcactggct cgtcggctga tgtcaacatc
      481 gttgagactg ctgaggctcg cgagaagcga atgagggatg aggagaaata tgctcgtgaa
      541 caggaggcca tcaaccgtca tgctgacaag gacctggaaa agaagacgga agcctaccgc
      601 aaggaggccg agcacgaggc tgagaagatc cgcaaggcgt tggagaagca acacgagcga
      661 gacatcgagt tccgcaagga agttgtggga agtaccatcg agaagcagaa ggcggaaatc
      721 gagctggaag ccaagcgagc aaaggccgct ttggagcacg aacgtcagct ggccaacgat
      781 gcgttggagc gttctaagat gcataccgat gtccaagtca cgatggacac tgcagccgga
      841 cataccgtca gcggtggaac cacgatgtcg tcatcggagc agcacagttc ttcgcacagc
      901 tcatctcgca ctggcggact gtagaattcg atatcaagct tatcgatgct agcactagtc
      961 ctaggctggt tccgcgtggg tccgagaacc tgtacttcca gggctcccct atactaggtt
     1021 attggaaaat taagggcctt gtgcaaccca ctcgacttct tttggaatat cttgaagaaa
     1081 aatatgaaga gcatttgtat gagcgcgatg aaggtgataa atggcgaaac aaaaagtttg
     1141 aattgggttt ggagtttccc aatcttcctt attatattga tggtgatgtt aaattaacac
     1201 agtctatggc catcatacgt tatatagctg acaagcacaa catgttgggt ggttgtccaa
     1261 aagagcgtgc agagatttca atgcttgaag gagcggtttt ggatattaga tacggtgttt
     1321 cgagaattgc atatagtaaa gactttgaaa ctctcaaagt tgattttctt agcaagctac
     1381 ctgaaatgct gaaaatgttc gaagatcgtt tatgtcataa aacatattta aatggtgatc
     1441 atgtaaccca tcctgacttc atgttgtatg acgctcttga tgttgtttta tacatggacc
     1501 caatgtgcct ggatgcgttc ccaaaattag tttgttttaa aaaacgtatt gaagctatcc
     1561 cacaaattga taagtacttg aaatccagca agtatatagc atggcctttg cagggctggc
     1621 aagccacgtt tggtggtggc gaccatcctc caaaataact cgagcaccac caccaccacc
     1681 actgagatcc ggctgctaac aaagcccgaa aggaagctga gttggctgct gccaccgctg
     1741 agcaataact agcataaccc cttggggcct ctaaacgggt cttgaggggt tttttgctga
     1801 aaggaggaac tatatccgga ttggcgaatg ggacgcgccc tgtagcggcg cattaagcgc
     1861 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
     1921 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     1981 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     2041 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     2101 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     2161 caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg
     2221 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct
     2281 tacaatttag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
     2341 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa
     2401 tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt
     2461 gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct
     2521 gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc
     2581 cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta
     2641 tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac
     2701 tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc
     2761 atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac
     2821 ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg
     2881 gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac
     2941 gagcgtgaca ccacgatgcc tgcagcaatg gcaacaacgt tgcgcaaact attaactggc
     3001 gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt
     3061 gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga
     3121 gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc
     3181 cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag
     3241 atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca
     3301 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc
     3361 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     3421 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     3481 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     3541 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt
     3601 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     3661 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     3721 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     3781 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     3841 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     3901 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     3961 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     4021 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     4081 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt
     4141 accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca
     4201 gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca tctgtgcggt
     4261 atttcacacc gcaatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
     4321 agtatacact ccgctatcgc tacgtgactg ggtcatggct gcgccccgac acccgccaac
     4381 acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt
     4441 gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag
     4501 gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc
     4561 atccgcgtcc agctcgttga gtttctccag aagcgttaat gtctggcttc tgataaagcg
     4621 ggccatgtta agggcggttt tttcctgttt ggtcactgat gcctccgtgt aagggggatt
     4681 tctgttcatg ggggtaatga taccgatgaa acgagagagg atgctcacga tacgggttac
     4741 tgatgatgaa catgcccggt tactggaacg ttgtgagggt aaacaactgg cggtatggat
     4801 gcggcgggac cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta atacagatgt
     4861 aggtgttcca cagggtagcc agcagcatcc tgcgatgcag atccggaaca taatggtgca
     4921 gggcgctgac ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga ccattcatgt
     4981 tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct cgcgtatcgg
     5041 tgattcattc tgctaaccag taaggcaacc ccgccagcct agccgggtcc tcaacgacag
     5101 gagcacgatc atgcgcaccc gtggggccgc catgccggcg ataatggcct gcttctcgcc
     5161 gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg agggcgtgca agattccgaa
     5221 taccgcaagc gacaggccga tcatcgtcgc gctccagcga aagcggtcct cgccgaaaat
     5281 gacccagagc gctgccggca cctgtcctac gagttgcatg ataaagaaga cagtcataag
     5341 tgcggcgacg atagtcatgc cccgcgccca ccggaaggag ctgactgggt tgaaggctct
     5401 caagggcatc ggtcgagatc ccggtgccta atgagtgagc taacttacat taattgcgtt
     5461 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     5521 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag ggtggttttt cttttcacca
     5581 gtgagacggg caacagctga ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc
     5641 ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt aacggcggga
     5701 tataacatga gctgtcttcg gtatcgtcgt atcccactac cgagatatcc gcaccaacgc
     5761 gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg ttggcaacca
     5821 gcatcgcagt gggaacgatg ccctcattca gcatttgcat ggtttgttga aaaccggaca
     5881 tggcactcca gtcgccttcc cgttccgcta tcggctgaat ttgattgcga gtgagatatt
     5941 tatgccagcc agccagacgc agacgcgccg agacagaact taatgggccc gctaacagcg
     6001 cgatttgctg gtgacccaat gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat
     6061 gggagaaaat aatactgttg atgggtgtct ggtcagagac atcaagaaat aacgccggaa
     6121 cattagtgca ggcagcttcc acagcaatgg catcctggtc atccagcgga tagttaatga
     6181 tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc
     6241 cgcttcgttc taccatcgac accaccacgc tggcacccag ttgatcggcg cgagatttaa
     6301 tcgccgcgac aatttgcgac ggcgcgtgca gggccagact ggaggtggca acgccaatca
     6361 gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg
     6421 ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac gtggctggcc tggttcacca
     6481 cgcgggaaac ggtctgataa gagacaccgg catactctgc gacatcgtat aacgttactg
     6541 gtttcacatt caccaccctg aattgactct cttccgggcg ctatcatgcc ataccgcgaa
     6601 aggttttgcg ccattcgatg gtgtccggga tctcgacgct ctcccttatg cgactcctgc
     6661 attaggaagc agcccagtag taggttgagg ccgttgagca ccgccgccgc aaggaatggt
     6721 gcatgcaagg agatggcgcc caacagtccc ccggccacgg ggcctgccac catacccacg
     6781 ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg
     6841 gcgatatagg cgccagcaac cgcacctgtg gcgccggtga tgccggccac gatgcgtccg
     6901 gcgtagagga tcgagatctc gatcccgcga aattaatacg actcactata ggggaattgt
     6961 gagcggataa caattcccct ctagaaataa ttttgtttaa ctttaagaag gagatata
//
