LOCUS       sequence-178997-        6586 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To express His-tagged MAHS in bacteria.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6586)
  AUTHORS   Tanaka S, Tanaka J, Miwa Y, Horikawa DD, Katayama T, Arakawa K, 
            Toyoda A, Kubo T, Kunieda T
  TITLE     Novel mitochondria-targeted heat-soluble proteins identified in the 
            anhydrobiotic Tardigrade improve osmotic tolerance of human cells.
  JOURNAL   PLoS One. 2015 Feb 12;10(2):e0118272. eCollection 2015.
  PUBMED    25675104
REFERENCE   2  (bases 1 to 6586)
  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: "2015-02-12"; volume: "10(2)"; pages: "e0118272. eCollection 
            2015"
COMMENT     Sequence Label: pEThT-MAHS delta MTP
FEATURES             Location/Qualifiers
     source          1..6586
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     18..37
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        18..36
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    37..61
                     /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             76..98
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             109..126
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             636..653
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             654..674
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             675..1328
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="SPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKF
                     ELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYG
                     VSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLY
                     MDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     CDS             1335..1352
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1405..1423)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1419..1466
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1503..1958
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1590..1609)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1800..1821
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1984..2088
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2089..2157,2158..2949)
                     /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(2307..2326)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3120..3708
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3609..3628
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3862..3879
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3894..4033
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3959..3978)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4119..4141
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4135..4326)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5135..6217)
                     /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     6179..6198
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6218..6295)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6501..6520
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 tctcgatccc gcgaaattaa tacgactcac tataggggaa ttgtgagcgg ataacaattc
       61 ccctctagaa ataattttgt ttaactttaa gaaggagata tacatatgca ccatcaccat
      121 caccatggat ccgtgcaaca acagttccga aacgtcactg gagtgaatgc cgctcctgtt
      181 gtagcctttg ataatggatc agttctatac agtgaaagaa tccactcgca gagttcgcag
      241 aagcaggccc cgactacagt accaacagga tccgtcagca attcccctca accggaagga
      301 aaggcaaacg aagctgctga acgcgcaaaa cagtttatga atcctccagt tgcgccaatg
      361 gatcctgtcg acaagaatga atttgtcgcc atgccggaga tgggtcgtag taatggaaat
      421 ggagaaaaca aacaagctgc tgatttcatg aaaaaccaag gtgacaccga tatggattcc
      481 cagtacgcgc ctgattcatc gaagaacacg aaatcggttc ccacgaagga aatcgttgct
      541 gaagatggtt cgatgagcat tgaggatatc aagaaagcta cgcaggttac tcctggagtt
      601 gcagttaaaa acgagggtgt ttagctagtc ctaggctggt tccgcgtggg tccgagaacc
      661 tgtacttcca gggctcccct atactaggtt attggaaaat taagggcctt gtgcaaccca
      721 ctcgacttct tttggaatat cttgaagaaa aatatgaaga gcatttgtat gagcgcgatg
      781 aaggtgataa atggcgaaac aaaaagtttg aattgggttt ggagtttccc aatcttcctt
      841 attatattga tggtgatgtt aaattaacac agtctatggc catcatacgt tatatagctg
      901 acaagcacaa catgttgggt ggttgtccaa aagagcgtgc agagatttca atgcttgaag
      961 gagcggtttt ggatattaga tacggtgttt cgagaattgc atatagtaaa gactttgaaa
     1021 ctctcaaagt tgattttctt agcaagctac ctgaaatgct gaaaatgttc gaagatcgtt
     1081 tatgtcataa aacatattta aatggtgatc atgtaaccca tcctgacttc atgttgtatg
     1141 acgctcttga tgttgtttta tacatggacc caatgtgcct ggatgcgttc ccaaaattag
     1201 tttgttttaa aaaacgtatt gaagctatcc cacaaattga taagtacttg aaatccagca
     1261 agtatatagc atggcctttg cagggctggc aagccacgtt tggtggtggc gaccatcctc
     1321 caaaataact cgagcaccac caccaccacc actgagatcc ggctgctaac aaagcccgaa
     1381 aggaagctga gttggctgct gccaccgctg agcaataact agcataaccc cttggggcct
     1441 ctaaacgggt cttgaggggt tttttgctga aaggaggaac tatatccgga ttggcgaatg
     1501 ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     1561 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     1621 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
     1681 tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg
     1741 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
     1801 tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt
     1861 ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt
     1921 taacgcgaat tttaacaaaa tattaacgct tacaatttag gtggcacttt tcggggaaat
     1981 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
     2041 agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa
     2101 catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac
     2161 ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac
     2221 atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt
     2281 ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc
     2341 gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca
     2401 ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc
     2461 ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag
     2521 gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa
     2581 ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgcagcaatg
     2641 gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa
     2701 ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg
     2761 gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt
     2821 gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt
     2881 caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag
     2941 cattggtaac tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat
     3001 ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct
     3061 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     3121 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     3181 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     3241 agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc
     3301 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     3361 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     3421 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     3481 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     3541 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     3601 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     3661 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     3721 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     3781 ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc
     3841 cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg
     3901 cggtattttc tccttacgca tctgtgcggt atttcacacc gcaatggtgc actctcagta
     3961 caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg
     4021 ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct
     4081 gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag
     4141 gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc
     4201 gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag
     4261 aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt
     4321 ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa
     4381 acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg
     4441 ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg
     4501 tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc
     4561 tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta
     4621 cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca
     4681 gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc
     4741 ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc
     4801 catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa
     4861 ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc
     4921 gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac
     4981 gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca
     5041 ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta
     5101 atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     5161 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     5221 tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca
     5281 ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa
     5341 aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt
     5401 atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg
     5461 cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca
     5521 gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta
     5581 tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg
     5641 agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat
     5701 gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct
     5761 ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg
     5821 catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat
     5881 tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc
     5941 tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca
     6001 gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg
     6061 ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt
     6121 tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg
     6181 catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct
     6241 cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga
     6301 tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg
     6361 ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc
     6421 ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg
     6481 cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg
     6541 gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgaga
//
