LOCUS       sequence-178988-        6809 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To express His-tagged CAHS1 in bacteria.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6809)
  AUTHORS   Yamaguchi A, Tanaka S, Yamaguchi S, Kuwahara H, Takamura C, 
            Imajoh-Ohmi S, Horikawa DD, Toyoda A, Katayama T, Arakawa K, 
            Fujiyama A, Kubo T, Kunieda T
  TITLE     Two novel heat-soluble protein families abundantly expressed in an 
            anhydrobiotic tardigrade.
  JOURNAL   PLoS One. 2012;7(8):e44209.
  PUBMED    22937162
REFERENCE   2  (bases 1 to 6809)
  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 One.";
            date: "2012"; volume: "7(8)"; pages: "e44209"
COMMENT     Sequence Label: pEThT-CAHS1
FEATURES             Location/Qualifiers
     source          1..6809
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     204..223
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     307..326
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        307..325
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    326..350
                     /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             365..387
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             398..415
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1148..1165
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1166..1186
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1187..1840
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="SPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKF
                     ELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYG
                     VSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLY
                     MDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     CDS             1847..1864
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1917..1935)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1931..1978
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2015..2470
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2102..2121)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2312..2333
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2496..2600
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2601..2669,2670..3461)
                     /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(2819..2838)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3632..4220
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4121..4140
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4374..4391
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4406..4545
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4471..4490)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4631..4653
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4647..4838)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5647..6729)
                     /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     6691..6710
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6730..6807)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 ggatctcgac gctctccctt atgcgactcc tgcattagga agcagcccag tagtaggttg
       61 aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc gcccaacagt
      121 cccccggcca cggggcctgc caccataccc acgccgaaac aagcgctcat gagcccgaag
      181 tggcgagccc gatcttcccc atcggtgatg tcggcgatat aggcgccagc aaccgcacct
      241 gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat ctcgatcccg
      301 cgaaattaat acgactcact ataggggaat tgtgagcgga taacaattcc cctctagaaa
      361 taattttgtt taactttaag aaggagatat acatatgcac catcaccatc accatggatc
      421 catgccttac gaaaagcacg ttgaacagac ggtggtggaa aaaactgagc agcctggaca
      481 ctcgtcgacg caccatgctc ccgcccaaag gaccgtagct cgcgagcagg aggaagttgt
      541 ccacaaagag tttacccata ccgacattcg agttccccac atcgacgcac ctcctccaat
      601 catcgctgcc agcgcagtgg gcttggccga ggagatcgtc agtcatggtt tccaagcctc
      661 ggcggcccgc atcagcggtg cttccaccga ggtcgatatg cgcccaagtc ccaagctagc
      721 cgaggaagct cgtcgtgatg ccgaacgata tcaaaaggag cacgagatga tcaacagaca
      781 agccgaagcc acgctgcaaa agaaggcgga ggagtaccgt caccagactg aggcggaagc
      841 cgagaagatt cgccgcgaac tggaaaagca gcatgaacgg gacatccagt tcaggaagga
      901 cctcatcgat cagaccatcg aaaagcagaa gcgcgaagtt gatctggaag ccaagatggc
      961 caaacgcgag ctggatcgcg aagcgcagtt ggctaaggaa gccctggaac gttctcgaat
     1021 ggccaccaac gtggaagtca cgctggatac cgcagcggga catacagtaa gtgggggaac
     1081 taccgtctcc agcgtcgaca aagttgaaac cgtccgcgag cgcaaacatc attaaactag
     1141 tcctaggctg gttccgcgtg ggtccgagaa cctgtacttc cagggctccc ctatactagg
     1201 ttattggaaa attaagggcc ttgtgcaacc cactcgactt cttttggaat atcttgaaga
     1261 aaaatatgaa gagcatttgt atgagcgcga tgaaggtgat aaatggcgaa acaaaaagtt
     1321 tgaattgggt ttggagtttc ccaatcttcc ttattatatt gatggtgatg ttaaattaac
     1381 acagtctatg gccatcatac gttatatagc tgacaagcac aacatgttgg gtggttgtcc
     1441 aaaagagcgt gcagagattt caatgcttga aggagcggtt ttggatatta gatacggtgt
     1501 ttcgagaatt gcatatagta aagactttga aactctcaaa gttgattttc ttagcaagct
     1561 acctgaaatg ctgaaaatgt tcgaagatcg tttatgtcat aaaacatatt taaatggtga
     1621 tcatgtaacc catcctgact tcatgttgta tgacgctctt gatgttgttt tatacatgga
     1681 cccaatgtgc ctggatgcgt tcccaaaatt agtttgtttt aaaaaacgta ttgaagctat
     1741 cccacaaatt gataagtact tgaaatccag caagtatata gcatggcctt tgcagggctg
     1801 gcaagccacg tttggtggtg gcgaccatcc tccaaaataa ctcgagcacc accaccacca
     1861 ccactgagat ccggctgcta acaaagcccg aaaggaagct gagttggctg ctgccaccgc
     1921 tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct
     1981 gaaaggagga actatatccg gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc
     2041 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
     2101 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     2161 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     2221 aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
     2281 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
     2341 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat
     2401 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg
     2461 cttacaattt aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     2521 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     2581 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
     2641 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
     2701 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
     2761 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
     2821 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
     2881 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
     2941 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
     3001 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
     3061 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
     3121 acgagcgtga caccacgatg cctgcagcaa tggcaacaac gttgcgcaaa ctattaactg
     3181 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
     3241 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
     3301 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
     3361 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     3421 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     3481 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     3541 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     3601 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     3661 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     3721 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc
     3781 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     3841 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     3901 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     3961 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     4021 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     4081 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     4141 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     4201 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     4261 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     4321 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     4381 cagtgagcga ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg
     4441 gtatttcaca ccgcaatggt gcactctcag tacaatctgc tctgatgccg catagttaag
     4501 ccagtataca ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg acacccgcca
     4561 acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct
     4621 gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg
     4681 aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt
     4741 tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta atgtctggct tctgataaag
     4801 cgggccatgt taagggcggt tttttcctgt ttggtcactg atgcctccgt gtaaggggga
     4861 tttctgttca tgggggtaat gataccgatg aaacgagaga ggatgctcac gatacgggtt
     4921 actgatgatg aacatgcccg gttactggaa cgttgtgagg gtaaacaact ggcggtatgg
     4981 atgcggcggg accagagaaa aatcactcag ggtcaatgcc agcgcttcgt taatacagat
     5041 gtaggtgttc cacagggtag ccagcagcat cctgcgatgc agatccggaa cataatggtg
     5101 cagggcgctg acttccgcgt ttccagactt tacgaaacac ggaaaccgaa gaccattcat
     5161 gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc
     5221 ggtgattcat tctgctaacc agtaaggcaa ccccgccagc ctagccgggt cctcaacgac
     5281 aggagcacga tcatgcgcac ccgtggggcc gccatgccgg cgataatggc ctgcttctcg
     5341 ccgaaacgtt tggtggcggg accagtgacg aaggcttgag cgagggcgtg caagattccg
     5401 aataccgcaa gcgacaggcc gatcatcgtc gcgctccagc gaaagcggtc ctcgccgaaa
     5461 atgacccaga gcgctgccgg cacctgtcct acgagttgca tgataaagaa gacagtcata
     5521 agtgcggcga cgatagtcat gccccgcgcc caccggaagg agctgactgg gttgaaggct
     5581 ctcaagggca tcggtcgaga tcccggtgcc taatgagtga gctaacttac attaattgcg
     5641 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     5701 ggccaacgcg cggggagagg cggtttgcgt attgggcgcc agggtggttt ttcttttcac
     5761 cagtgagacg ggcaacagct gattgccctt caccgcctgg ccctgagaga gttgcagcaa
     5821 gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg
     5881 gatataacat gagctgtctt cggtatcgtc gtatcccact accgagatat ccgcaccaac
     5941 gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc gccatctgat cgttggcaac
     6001 cagcatcgca gtgggaacga tgccctcatt cagcatttgc atggtttgtt gaaaaccgga
     6061 catggcactc cagtcgcctt cccgttccgc tatcggctga atttgattgc gagtgagata
     6121 tttatgccag ccagccagac gcagacgcgc cgagacagaa cttaatgggc ccgctaacag
     6181 cgcgatttgc tggtgaccca atgcgaccag atgctccacg cccagtcgcg taccgtcttc
     6241 atgggagaaa ataatactgt tgatgggtgt ctggtcagag acatcaagaa ataacgccgg
     6301 aacattagtg caggcagctt ccacagcaat ggcatcctgg tcatccagcg gatagttaat
     6361 gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac aggcttcgac
     6421 gccgcttcgt tctaccatcg acaccaccac gctggcaccc agttgatcgg cgcgagattt
     6481 aatcgccgcg acaatttgcg acggcgcgtg cagggccaga ctggaggtgg caacgccaat
     6541 cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt aattcagctc
     6601 cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa acgtggctgg cctggttcac
     6661 cacgcgggaa acggtctgat aagagacacc ggcatactct gcgacatcgt ataacgttac
     6721 tggtttcaca ttcaccaccc tgaattgact ctcttccggg cgctatcatg ccataccgcg
     6781 aaaggttttg cgccattcga tggtgtccg
//
