LOCUS       sequence-177698-        5694 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To express His-tagged Dsup in bacteria.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5694)
  AUTHORS   Hashimoto T, Horikawa DD, Saito Y, Kuwahara H, Kozuka-Hata H, Shin-I
            T, Minakuchi Y, Ohishi K, Motoyama A, Aizu T, Enomoto A, Kondo K, 
            Tanaka S, Hara Y, Koshikawa S, Sagara H, Miura T, Yokobori S, 
            Miyagawa K, Suzuki Y, Kubo T, Oyama M, Kohara Y, Fujiyama A, Arakawa
            K, Katayama T, Toyoda A, Kunieda T
  TITLE     Extremotolerant tardigrade genome and improved radiotolerance of 
            human cultured cells by tardigrade-unique protein.
  JOURNAL   Nat Commun. 2016 Sep 20;7:12808. doi: 10.1038/ncomms12808.
  PUBMED    27649274
REFERENCE   2  (bases 1 to 5694)
  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: "Nat 
            Commun."; date: "2016-09-20"; volume: "7:12808. doi"; pages: " 
            10.1038/ncomms12808"
COMMENT     Sequence Label: pCold-I-Dsup
FEATURES             Location/Qualifiers
     source          1..5694
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     411..430
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(682..1764)
                     /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     1726..1745
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1765..1842)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        1942..2008
                     /gene="cspA"
                     /label=cspA promoter
                     /note="promoter of the E. coli cold shock protein cspA gene
                     (Mitta et al., 1997)"
     protein_bind    2011..2027
                     /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)."
     5'UTR           2049..2182
                     /gene="cspA"
                     /label=cspA 5'UTR
                     /note="5'UTR of the E. coli cold shock protein cspA gene 
                     (Mitta et al., 1997)"
     CDS             2183..2197
                     /codon_start=1
                     /gene="cspA"
                     /product="translation enhancing element for E. coli (Qing 
                     et al., 2004)"
                     /label=TEE
                     /translation="MNHKV"
     CDS             2198..2215
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2216..2227
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     3'UTR           3582..3726
                     /gene="cspA"
                     /label=cspA 3'UTR
                     /note="3'UTR of the E. coli cold shock protein cspA gene 
                     (Mitta et al., 1997)"
     primer_bind     complement(3767..3789)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3889..3908
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(3925..4380)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4062..4083)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4274..4293
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4480..4584
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4585..4653,4654..5445)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(4803..4822)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(5616..5694,1..510)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
       61 aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
      121 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
      181 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
      241 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
      301 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
      361 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
      421 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
      481 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
      541 ctggcctttt gctggccttt tgctcacata gtcatgcccc gcgcccaccg gaaggagctg
      601 actgggttga aggctctcaa gggcatcggt cgagatcccg gtgcctaatg agtgagctaa
      661 cttacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
      721 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt
      781 ggtttttctt ttcaccagtg agacgggcaa cagctgattg cccttcaccg cctggccctg
      841 agagagttgc agcaagcggt ccacgctggt ttgccccagc aggcgaaaat cctgtttgat
      901 ggtggttaac ggcgggatat aacatgagct gtcttcggta tcgtcgtatc ccactaccga
      961 gatatccgca ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc ccagcgccat
     1021 ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc tcattcagca tttgcatggt
     1081 ttgttgaaaa ccggacatgg cactccagtc gccttcccgt tccgctatcg gctgaatttg
     1141 attgcgagtg agatatttat gccagccagc cagacgcaga cgcgccgaga cagaacttaa
     1201 tgggcccgct aacagcgcga tttgctggtg acccaatgcg accagatgct ccacgcccag
     1261 tcgcgtaccg tcttcatggg agaaaataat actgttgatg ggtgtctggt cagagacatc
     1321 aagaaataac gccggaacat tagtgcaggc agcttccaca gcaatggcat cctggtcatc
     1381 cagcggatag ttaatgatca gcccactgac gcgttgcgcg agaagattgt gcaccgccgc
     1441 tttacaggct tcgacgccgc ttcgttctac catcgacacc accacgctgg cacccagttg
     1501 atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga
     1561 ggtggcaacg ccaatcagca acgactgttt gcccgccagt tgttgtgcca cgcggttggg
     1621 aatgtaattc agctccgcca tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg
     1681 gctggcctgg ttcaccacgc gggaaacggt ctgataagag acaccggcat actctgcgac
     1741 atcgtataac gttactggtt tcacattcac caccctgaat tgactctctt ccgggcgcta
     1801 tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg tccgggatct cgacgctctc
     1861 ccttatgcga ctcctgcatt aggaagcagc ccagtagtag gttgaggccg ttgagcaccg
     1921 ccgccgcaag gaatggtgtg gccgattaat cataaatatg aaaaataatt gttgcatcac
     1981 ccgccaatgc gtggcttaat gcacatcaaa ttgtgagcgg ataacaattt gatgtgctag
     2041 cgcatatcca gtgtagtaag gcaagtccct tcaagagtta tcgttgatac ccctcgtagt
     2101 gcacattcct ttaacgcttc aaaatctgta aagcacgcca tatcgccgaa aggcacactt
     2161 aattattaag aggtaataca ccatgaatca caaagtgcat catcatcatc atcatatcga
     2221 aggtaggcat atggcatcca cacaccaatc atccacagaa ccctcttcca caggtaaatc
     2281 tgaggaaacg aagaaagatg cttcgcaagg gagcgggcaa gactccaaga acgtaaccgt
     2341 taccaaaggt accggttcct ccgccacctc agctgccatt gtcaagacag gaggatccca
     2401 aggcaaagat tcctctacta cagcgggctc ttctagtact cagggacaga agttcagtac
     2461 tacacctacc gacccgaaaa ctttcagctc tgaccaaaag gagaaatcca aaagcccagc
     2521 caaagaagtc ccgtctggtg gcgatagtaa gtcccaaggt gacaccaagt ctcaaagcga
     2581 cgccaaatct tctggacaaa gtcagggcca gtctaaagac agcggcaaat catcttccga
     2641 cagtagcaag agtcactctg tcatcggagc tgtcaaagac gtcgttgcag gcgccaaaga
     2701 tgtcgcagga aaagccgtcg aggatgctcc tagcatcatg catactgcag tcgatgctgt
     2761 gaagaacgca gccacgactg tgaaggatgt ggcatcgtcg gctgcatcga ctgtggcgga
     2821 gaaggtagtc gatgcttacc acagtgtggt gggagacaag acggacgaca agaaagaggg
     2881 cgagcacagc ggcgacaaga aggacgactc caaagctgga agtggctctg gacaaggtgg
     2941 tgacaacaag aagtctgaag gagagacttc tggccaagca gaatccagct ctggcaacga
     3001 aggagctgct ccagccaaag gccgtggtcg tggacggcct ccagcagctg ctaaaggagt
     3061 tgctaagggt gctgcaaagg gcgctgccgc ctccaaagga gccaagagcg gtgctgaatc
     3121 ctccaaggga ggagaacagt cgtcaggaga tatcgagatg gcagatgctt cctccaaggg
     3181 aggctcggac cagagggatt ccgcggcgac cgttggcgaa ggtggtgcat caggcagtga
     3241 gggtggagct aagaaaggca gagggcgggg cgctggtaag aaagcggatg cgggtgatac
     3301 gtccgctgag ccgcctcggc ggtcgtcccg cctgacgtct tcaggtacag gggcgggttc
     3361 cgctccagct gcagcgaaag gcggagcgaa gcgtgctgct tcttcctcca gtacaccttc
     3421 caacgctaag aagcaagcga ctggaggtgc tggcaaagct gctgccacca aagcaactgc
     3481 tgccaaatcg gcagcctcta aagctcccca gaatggcgca ggtgccaaga agaagggagg
     3541 aaaggctgga ggacggaaga ggaagtaatc tagataggta atctctgctt aaaagcacag
     3601 aatctaagat ccctgccatt tggcggggat ttttttattt gttttcagga aataaataat
     3661 cgatcgcgta ataaaatcta ttattatttt tgtgaagaat aaatttgggt gcaatgagaa
     3721 tgcgcaggcc ctttcgtctc gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc
     3781 agctcccgga gacggtcaca gcttgtctgt aagcggatgc cgggagcaga caagcccgtc
     3841 agggcgcgtc agcgggtgtt ggcgggtgtc ggggctggct taactatgcg gcatcagagc
     3901 agattgtact gagagtgcac cataaaattg taaacgttaa tattttgtta aaattcgcgt
     3961 taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt
     4021 ataaatcaaa agaatagccc gagatagggt tgagtgttgt tccagtttgg aacaagagtc
     4081 cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg
     4141 gcccactacg tgaaccatca cccaaatcaa gttttttggg gtcgaggtgc cgtaaagcac
     4201 taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg
     4261 tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag
     4321 cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt
     4381 actatggttg ctttgacgta tgcggtgtga aataccgcac agatgcgtaa ggagaaaata
     4441 ccgcatcagg cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
     4501 tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt
     4561 caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc
     4621 ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa
     4681 gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt
     4741 aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt
     4801 ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc
     4861 atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg
     4921 gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg
     4981 gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac
     5041 atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca
     5101 aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta
     5161 actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat
     5221 aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa
     5281 tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag
     5341 ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat
     5401 agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt
     5461 tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg
     5521 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga
     5581 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     5641 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccg
//
