LOCUS       sequence-178998-        6976 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To express His-tagged RvLEAM in bacteria.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6976)
  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 6976)
  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-RvLEAM
FEATURES             Location/Qualifiers
     source          1..6976
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     200..219
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     453..470
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    485..624
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(550..569)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     710..732
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(726..917)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1726..2808)
                     /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     2770..2789
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2809..2886)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     3092..3111
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     3195..3214
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3195..3213
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3214..3238
                     /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             3253..3275
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3286..3303
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             4203..4220
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             4221..4241
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             4242..4895
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="SPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKF
                     ELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYG
                     VSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLY
                     MDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     CDS             4902..4919
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4972..4990)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4986..5033
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      5070..5525
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5157..5176)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5367..5388
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5551..5655
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5656..5724,5725..6516)
                     /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(5874..5893)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(6687..6976,1..299)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
       61 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
      121 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
      181 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
      241 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
      301 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
      361 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg
      421 ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag
      481 agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcaatggtg
      541 cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg
      601 ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga
      661 cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc
      721 atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca
      781 tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg
      841 agtttctcca gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt
      901 ttttcctgtt tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg
      961 ataccgatga aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg
     1021 ttactggaac gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa
     1081 atcactcagg gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc
     1141 cagcagcatc ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt
     1201 tccagacttt acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac
     1261 gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca
     1321 gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc
     1381 cgtggggccg ccatgccggc gataatggcc tgcttctcgc cgaaacgttt ggtggcggga
     1441 ccagtgacga aggcttgagc gagggcgtgc aagattccga ataccgcaag cgacaggccg
     1501 atcatcgtcg cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc
     1561 acctgtccta cgagttgcat gataaagaag acagtcataa gtgcggcgac gatagtcatg
     1621 ccccgcgccc accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat
     1681 cccggtgcct aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc
     1741 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     1801 ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg
     1861 attgcccttc accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc
     1921 cagcaggcga aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc
     1981 ggtatcgtcg tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat
     2041 ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat
     2101 gccctcattc agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc
     2161 ccgttccgct atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg
     2221 cagacgcgcc gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa
     2281 tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt
     2341 gatgggtgtc tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc
     2401 cacagcaatg gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg
     2461 cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga
     2521 caccaccacg ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga
     2581 cggcgcgtgc agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc
     2641 cagttgttgt gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt
     2701 ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata
     2761 agagacaccg gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct
     2821 gaattgactc tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat
     2881 ggtgtccggg atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta
     2941 gtaggttgag gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc
     3001 ccaacagtcc cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga
     3061 gcccgaagtg gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa
     3121 ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atcgagatct
     3181 cgatcccgcg aaattaatac gactcactat aggggaattg tgagcggata acaattcccc
     3241 tctagaaata attttgttta actttaagaa ggagatatac atatgcacca tcaccatcac
     3301 catggatcca tgtttctcgc ccgaaacgct ggacgcgctg gatatcgagg tgttgtagct
     3361 taccagcaag cagcctcttt ctctgtttca tcagctaagg ctgctggatc acggtcaagc
     3421 ggtggctccg acgctggcga ttatgctagg gaggccgcgg aacatgccaa agctggattg
     3481 aaggatctca agaatgaagc ctcgtggaaa gccaaaggtg tcgctaacca agccgctgga
     3541 gcattcgaac gagcgaagga tacagtgaag gaaggtgtcc atgacatgaa gcgcagtgga
     3601 agccgcgtct tcgaacaagg acaggaggaa gtagaggctg gtgctcaaca cgcaaaggct
     3661 ggctatcaga gtgccaagaa tgctgcgcaa gacactgccg caactcttaa agacaaagcc
     3721 ggcagcgcct ggaatcaagc gaaacacgtt gtggaagata agggagagga tgttgttgaa
     3781 gcagtcaagg acactgcttc aaaagtctgg ggcaaagcca aacatgtggc cgaggacgtt
     3841 aaggagaacg cgcaatcacc aggtggcatt gcggataagg cctccgatgt gtggtcagcg
     3901 gccaaggata aagctgctga tgtcttgtcg ggagctaagc acactgctga gaacctagct
     3961 cacaaagccc aagccgccat ccacgatgct acagcttctt caggctcgca gtcgcagtcg
     4021 cagtcccagt ctcagtatcg acaaggacaa caacagggaa gacaagatca gcagcaaagc
     4081 aagagccagt ggggacagac tagcccgcag tctccggatg gattccgccc tcaagccggc
     4141 cagggaccac aaggaggcaa gggacctggt caagctggcg gtcgacgata gctagtccta
     4201 ggctggttcc gcgtgggtcc gagaacctgt acttccaggg ctcccctata ctaggttatt
     4261 ggaaaattaa gggccttgtg caacccactc gacttctttt ggaatatctt gaagaaaaat
     4321 atgaagagca tttgtatgag cgcgatgaag gtgataaatg gcgaaacaaa aagtttgaat
     4381 tgggtttgga gtttcccaat cttccttatt atattgatgg tgatgttaaa ttaacacagt
     4441 ctatggccat catacgttat atagctgaca agcacaacat gttgggtggt tgtccaaaag
     4501 agcgtgcaga gatttcaatg cttgaaggag cggttttgga tattagatac ggtgtttcga
     4561 gaattgcata tagtaaagac tttgaaactc tcaaagttga ttttcttagc aagctacctg
     4621 aaatgctgaa aatgttcgaa gatcgtttat gtcataaaac atatttaaat ggtgatcatg
     4681 taacccatcc tgacttcatg ttgtatgacg ctcttgatgt tgttttatac atggacccaa
     4741 tgtgcctgga tgcgttccca aaattagttt gttttaaaaa acgtattgaa gctatcccac
     4801 aaattgataa gtacttgaaa tccagcaagt atatagcatg gcctttgcag ggctggcaag
     4861 ccacgtttgg tggtggcgac catcctccaa aataactcga gcaccaccac caccaccact
     4921 gagatccggc tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc accgctgagc
     4981 aataactagc ataacccctt ggggcctcta aacgggtctt gaggggtttt ttgctgaaag
     5041 gaggaactat atccggattg gcgaatggga cgcgccctgt agcggcgcat taagcgcggc
     5101 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
     5161 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     5221 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     5281 tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     5341 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     5401 ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt
     5461 aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgcttac
     5521 aatttaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     5581 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
     5641 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
     5701 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa
     5761 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
     5821 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt
     5881 ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat
     5941 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
     6001 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
     6061 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
     6121 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
     6181 cgtgacacca cgatgcctgc agcaatggca acaacgttgc gcaaactatt aactggcgaa
     6241 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
     6301 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
     6361 ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
     6421 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
     6481 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
     6541 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     6601 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     6661 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     6721 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     6781 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta
     6841 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     6901 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     6961 gactcaagac gatagt
//
