LOCUS       sequence-178994-        7007 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To express His-tagged CAHS3 in bacteria.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7007)
  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 7007)
  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-CAHS3
FEATURES             Location/Qualifiers
     source          1..7007
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      134..722
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     623..642
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     876..893
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    908..1047
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(973..992)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1133..1155
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1149..1340)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(2149..3231)
                     /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     3193..3212
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3232..3309)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     3515..3534
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     3618..3637
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3618..3636
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3637..3661
                     /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             3676..3698
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3709..3726
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             4657..4674
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             4675..4695
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             4696..5349
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="SPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKF
                     ELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYG
                     VSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLY
                     MDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     CDS             5356..5373
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5426..5444)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5440..5487
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      5524..5979
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5611..5630)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5821..5842
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        6005..6109
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6110..6178,6179..6970)
                     /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(6328..6347)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
       61 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
      121 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
      181 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
      241 aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt
      301 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
      361 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
      421 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
      481 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
      541 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
      601 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
      661 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
      721 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
      781 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag
      841 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg
      901 aagagcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcaatg
      961 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtata cactccgcta
     1021 tcgctacgtg actgggtcat ggctgcgccc cgacacccgc caacacccgc tgacgcgccc
     1081 tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc
     1141 tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc
     1201 tcatcagcgt ggtcgtgaag cgattcacag atgtctgcct gttcatccgc gtccagctcg
     1261 ttgagtttct ccagaagcgt taatgtctgg cttctgataa agcgggccat gttaagggcg
     1321 gttttttcct gtttggtcac tgatgcctcc gtgtaagggg gatttctgtt catgggggta
     1381 atgataccga tgaaacgaga gaggatgctc acgatacggg ttactgatga tgaacatgcc
     1441 cggttactgg aacgttgtga gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga
     1501 aaaatcactc agggtcaatg ccagcgcttc gttaatacag atgtaggtgt tccacagggt
     1561 agccagcagc atcctgcgat gcagatccgg aacataatgg tgcagggcgc tgacttccgc
     1621 gtttccagac tttacgaaac acggaaaccg aagaccattc atgttgttgc tcaggtcgca
     1681 gacgttttgc agcagcagtc gcttcacgtt cgctcgcgta tcggtgattc attctgctaa
     1741 ccagtaaggc aaccccgcca gcctagccgg gtcctcaacg acaggagcac gatcatgcgc
     1801 acccgtgggg ccgccatgcc ggcgataatg gcctgcttct cgccgaaacg tttggtggcg
     1861 ggaccagtga cgaaggcttg agcgagggcg tgcaagattc cgaataccgc aagcgacagg
     1921 ccgatcatcg tcgcgctcca gcgaaagcgg tcctcgccga aaatgaccca gagcgctgcc
     1981 ggcacctgtc ctacgagttg catgataaag aagacagtca taagtgcggc gacgatagtc
     2041 atgccccgcg cccaccggaa ggagctgact gggttgaagg ctctcaaggg catcggtcga
     2101 gatcccggtg cctaatgagt gagctaactt acattaattg cgttgcgctc actgcccgct
     2161 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
     2221 ggcggtttgc gtattgggcg ccagggtggt ttttcttttc accagtgaga cgggcaacag
     2281 ctgattgccc ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg
     2341 ccccagcagg cgaaaatcct gtttgatggt ggttaacggc gggatataac atgagctgtc
     2401 ttcggtatcg tcgtatccca ctaccgagat atccgcacca acgcgcagcc cggactcggt
     2461 aatggcgcgc attgcgccca gcgccatctg atcgttggca accagcatcg cagtgggaac
     2521 gatgccctca ttcagcattt gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc
     2581 ttcccgttcc gctatcggct gaatttgatt gcgagtgaga tatttatgcc agccagccag
     2641 acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt gctggtgacc
     2701 caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga aaataatact
     2761 gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag tgcaggcagc
     2821 ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc cactgacgcg
     2881 ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc gttctaccat
     2941 cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg
     3001 cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg actgtttgcc
     3061 cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg ccgcttccac
     3121 tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg aaacggtctg
     3181 ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca cattcaccac
     3241 cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt tgcgccattc
     3301 gatggtgtcc gggatctcga cgctctccct tatgcgactc ctgcattagg aagcagccca
     3361 gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg
     3421 cgcccaacag tcccccggcc acggggcctg ccaccatacc cacgccgaaa caagcgctca
     3481 tgagcccgaa gtggcgagcc cgatcttccc catcggtgat gtcggcgata taggcgccag
     3541 caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga
     3601 tctcgatccc gcgaaattaa tacgactcac tataggggaa ttgtgagcgg ataacaattc
     3661 ccctctagaa ataattttgt ttaactttaa gaaggagata tacatatgca ccatcaccat
     3721 caccatggat ccatgtcttc ccgacagaac cagcaatcgt ccagccaaca ctcgtcctcc
     3781 agccagcaag gtggtcaagg tggtcaaggt gttcaaggaa gttccagcta ctcgcgcacc
     3841 gaggtccaca ccagcagtgg aggacctacc atcggtggag cccagcgaac tgtccccgtc
     3901 ccccctggat ctcactccga ggtccatgag gagcgtgagg tcatcaagca tggtaccaaa
     3961 accgaaagcg agacccacgt cgtcaccgtc ccagtgacaa ctttcggcag caccaacatg
     4021 gaatctgtcc gaaccggctt caccgtcacc caagacaaga acttgaccgt tgctgctccc
     4081 aacatcgctg ctcccatcca cagcaacctc gaccttaacc tcggcggtgg agctcgcgct
     4141 gaaattaccg cagggaccac cgttgacttg agcaagatcc agcgcaagga tttgggacct
     4201 gaagagtatg ctcgctacaa ggccaaggtc gagcaactgg ccaggcaaga tgagcaagac
     4261 gcgggtatgc gcgctgccca gtaccgagag gaagtggagc gtgatgccga actcatccga
     4321 cagatcttgg agcgacaaca catccgtgat cttgaattcc gcaaggaaat ggttgagaac
     4381 caagtcaacc gacaagagag agaaatccag ctggaggctg agtacgcaat gcgagccctc
     4441 gagcttgagc gcaatgccgc taaagaggct ttggaaagcg ccaaggccca gactaacgtc
     4501 aatgtcaagg tcgagtccgc tattggtacc accgtctcca agggtgcaat ccagacttcc
     4561 gccgacaaga gcagcaccac caagactgga cccaccaccg tcactcagat taaacatacc
     4621 gaacaacaca ctgaacgccg atagactagt cctaggctgg ttccgcgtgg gtccgagaac
     4681 ctgtacttcc agggctcccc tatactaggt tattggaaaa ttaagggcct tgtgcaaccc
     4741 actcgacttc ttttggaata tcttgaagaa aaatatgaag agcatttgta tgagcgcgat
     4801 gaaggtgata aatggcgaaa caaaaagttt gaattgggtt tggagtttcc caatcttcct
     4861 tattatattg atggtgatgt taaattaaca cagtctatgg ccatcatacg ttatatagct
     4921 gacaagcaca acatgttggg tggttgtcca aaagagcgtg cagagatttc aatgcttgaa
     4981 ggagcggttt tggatattag atacggtgtt tcgagaattg catatagtaa agactttgaa
     5041 actctcaaag ttgattttct tagcaagcta cctgaaatgc tgaaaatgtt cgaagatcgt
     5101 ttatgtcata aaacatattt aaatggtgat catgtaaccc atcctgactt catgttgtat
     5161 gacgctcttg atgttgtttt atacatggac ccaatgtgcc tggatgcgtt cccaaaatta
     5221 gtttgtttta aaaaacgtat tgaagctatc ccacaaattg ataagtactt gaaatccagc
     5281 aagtatatag catggccttt gcagggctgg caagccacgt ttggtggtgg cgaccatcct
     5341 ccaaaataac tcgagcacca ccaccaccac cactgagatc cggctgctaa caaagcccga
     5401 aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc
     5461 tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg attggcgaat
     5521 gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     5581 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
     5641 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     5701 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
     5761 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     5821 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
     5881 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
     5941 ttaacgcgaa ttttaacaaa atattaacgc ttacaattta ggtggcactt ttcggggaaa
     6001 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     6061 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca
     6121 acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca
     6181 cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta
     6241 catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt
     6301 tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc
     6361 cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc
     6421 accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc
     6481 cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa
     6541 ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga
     6601 accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgcagcaat
     6661 ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca
     6721 attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc
     6781 ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat
     6841 tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag
     6901 tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa
     6961 gcattggtaa ctgtcagacc aagtttactc atatatactt tagattg
//
