LOCUS       sequence-178884-        6566 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To express His-tagged SAHS2 in bacteria.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6566)
  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 6566)
  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-SAHS2
FEATURES             Location/Qualifiers
     source          1..6566
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             607..624
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(677..695)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      691..738
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      775..1230
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(862..881)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1072..1093
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1256..1360
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1361..1429,1430..2221)
                     /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(1579..1598)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2392..2980
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2881..2900
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3134..3151
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3166..3305
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3231..3250)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3391..3413
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3407..3598)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4407..5489)
                     /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     5451..5470
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5490..5567)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5773..5792
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5876..5895
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5876..5894
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5895..5919
                     /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             5934..5956
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5967..5984
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             6065..6076
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             6474..6491
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             6492..6512
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             join(6513..6566,1..600)
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="SPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKF
                     ELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYG
                     VSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLY
                     MDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
ORIGIN
        1 cttttggaat atcttgaaga aaaatatgaa gagcatttgt atgagcgcga tgaaggtgat
       61 aaatggcgaa acaaaaagtt tgaattgggt ttggagtttc ccaatcttcc ttattatatt
      121 gatggtgatg ttaaattaac acagtctatg gccatcatac gttatatagc tgacaagcac
      181 aacatgttgg gtggttgtcc aaaagagcgt gcagagattt caatgcttga aggagcggtt
      241 ttggatatta gatacggtgt ttcgagaatt gcatatagta aagactttga aactctcaaa
      301 gttgattttc ttagcaagct acctgaaatg ctgaaaatgt tcgaagatcg tttatgtcat
      361 aaaacatatt taaatggtga tcatgtaacc catcctgact tcatgttgta tgacgctctt
      421 gatgttgttt tatacatgga cccaatgtgc ctggatgcgt tcccaaaatt agtttgtttt
      481 aaaaaacgta ttgaagctat cccacaaatt gataagtact tgaaatccag caagtatata
      541 gcatggcctt tgcagggctg gcaagccacg tttggtggtg gcgaccatcc tccaaaataa
      601 ctcgagcacc accaccacca ccactgagat ccggctgcta acaaagcccg aaaggaagct
      661 gagttggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg
      721 gtcttgaggg gttttttgct gaaaggagga actatatccg gattggcgaa tgggacgcgc
      781 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
      841 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
      901 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
      961 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc
     1021 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
     1081 tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga
     1141 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
     1201 attttaacaa aatattaacg cttacaattt aggtggcact tttcggggaa atgtgcgcgg
     1261 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
     1321 accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg
     1381 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac
     1441 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact
     1501 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat
     1561 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga
     1621 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac
     1681 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat
     1741 gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac
     1801 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct
     1861 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgcagcaa tggcaacaac
     1921 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga
     1981 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg
     2041 gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact
     2101 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac
     2161 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta
     2221 actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt
     2281 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
     2341 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     2401 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     2461 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     2521 gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     2581 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     2641 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     2701 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     2761 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     2821 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     2881 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     2941 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     3001 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
     3061 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg
     3121 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga tgcggtattt
     3181 tctccttacg catctgtgcg gtatttcaca ccgcaatggt gcactctcag tacaatctgc
     3241 tctgatgccg catagttaag ccagtataca ctccgctatc gctacgtgac tgggtcatgg
     3301 ctgcgccccg acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg
     3361 catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac
     3421 cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg
     3481 attcacagat gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta
     3541 atgtctggct tctgataaag cgggccatgt taagggcggt tttttcctgt ttggtcactg
     3601 atgcctccgt gtaaggggga tttctgttca tgggggtaat gataccgatg aaacgagaga
     3661 ggatgctcac gatacgggtt actgatgatg aacatgcccg gttactggaa cgttgtgagg
     3721 gtaaacaact ggcggtatgg atgcggcggg accagagaaa aatcactcag ggtcaatgcc
     3781 agcgcttcgt taatacagat gtaggtgttc cacagggtag ccagcagcat cctgcgatgc
     3841 agatccggaa cataatggtg cagggcgctg acttccgcgt ttccagactt tacgaaacac
     3901 ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc
     3961 ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa ccccgccagc
     4021 ctagccgggt cctcaacgac aggagcacga tcatgcgcac ccgtggggcc gccatgccgg
     4081 cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg accagtgacg aaggcttgag
     4141 cgagggcgtg caagattccg aataccgcaa gcgacaggcc gatcatcgtc gcgctccagc
     4201 gaaagcggtc ctcgccgaaa atgacccaga gcgctgccgg cacctgtcct acgagttgca
     4261 tgataaagaa gacagtcata agtgcggcga cgatagtcat gccccgcgcc caccggaagg
     4321 agctgactgg gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc taatgagtga
     4381 gctaacttac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     4441 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgcc
     4501 agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt caccgcctgg
     4561 ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt
     4621 ttgatggtgg ttaacggcgg gatataacat gagctgtctt cggtatcgtc gtatcccact
     4681 accgagatat ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc
     4741 gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt cagcatttgc
     4801 atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc tatcggctga
     4861 atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc cgagacagaa
     4921 cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag atgctccacg
     4981 cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt ctggtcagag
     5041 acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat ggcatcctgg
     5101 tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc
     5161 gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac gctggcaccc
     5221 agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg cagggccaga
     5281 ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg
     5341 ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa
     5401 acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc ggcatactct
     5461 gcgacatcgt ataacgttac tggtttcaca ttcaccaccc tgaattgact ctcttccggg
     5521 cgctatcatg ccataccgcg aaaggttttg cgccattcga tggtgtccgg gatctcgacg
     5581 ctctccctta tgcgactcct gcattaggaa gcagcccagt agtaggttga ggccgttgag
     5641 caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc ccccggccac
     5701 ggggcctgcc accataccca cgccgaaaca agcgctcatg agcccgaagt ggcgagcccg
     5761 atcttcccca tcggtgatgt cggcgatata ggcgccagca accgcacctg tggcgccggt
     5821 gatgccggcc acgatgcgtc cggcgtagag gatcgagatc tcgatcccgc gaaattaata
     5881 cgactcacta taggggaatt gtgagcggat aacaattccc ctctagaaat aattttgttt
     5941 aactttaaga aggagatata catatgcacc atcaccatca ccatggatct gctgatgacg
     6001 ctgctcacga agaaggcgta gaatggactg ggaaaccgtg gatgggcaaa tgggaatccg
     6061 acccatcgaa ggacgagaac gttgaggaat tcaaaaagaa gctccagctt ccgatgagcc
     6121 actcggaaat gaacaaaaac tccaaagttt ggatccatca ctacaagaag ggagacgagt
     6181 accatcacaa aatcatcatc aacgacgccc attacaaaaa cgatatcgtc ttcaagctgg
     6241 gtcaagagtc cgccggttcg tataacggct catctttcag cgtgaagtac gaggacaaag
     6301 acggcgctct agtcggaagc gtccactaca ctggcaccaa agaacagtct cttgacaaga
     6361 ccatcaacaa cgtcttcaag ctcgaaggtg accatctggt taagacttcc accatcgagg
     6421 gagtgaccat gaagcgccac tacaacaaac gccagtgaat cactagtcct aggctggttc
     6481 cgcgtgggtc cgagaacctg tacttccagg gctcccctat actaggttat tggaaaatta
     6541 agggccttgt gcaacccact cgactt
//
