LOCUS       sequence-174470-        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"
     promoter        15..81
                     /gene="cspA"
                     /label=cspA promoter
                     /note="promoter of the E. coli cold shock protein cspA gene
                     (Mitta et al., 1997)"
     protein_bind    84..100
                     /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           122..255
                     /gene="cspA"
                     /label=cspA 5'UTR
                     /note="5'UTR of the E. coli cold shock protein cspA gene 
                     (Mitta et al., 1997)"
     CDS             256..270
                     /codon_start=1
                     /gene="cspA"
                     /product="translation enhancing element for E. coli (Qing 
                     et al., 2004)"
                     /label=TEE
                     /translation="MNHKV"
     CDS             271..288
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             289..300
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     3'UTR           1655..1799
                     /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(1840..1862)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     1962..1981
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(1998..2453)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2135..2156)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2347..2366
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2553..2657
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2658..2726,2727..3518)
                     /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(2876..2895)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3689..4277
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4178..4197
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(4449..5531)
                     /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     5493..5512
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5532..5609)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 aaggaatggt gtggccgatt aatcataaat atgaaaaata attgttgcat cacccgccaa
       61 tgcgtggctt aatgcacatc aaattgtgag cggataacaa tttgatgtgc tagcgcatat
      121 ccagtgtagt aaggcaagtc ccttcaagag ttatcgttga tacccctcgt agtgcacatt
      181 cctttaacgc ttcaaaatct gtaaagcacg ccatatcgcc gaaaggcaca cttaattatt
      241 aagaggtaat acaccatgaa tcacaaagtg catcatcatc atcatcatat cgaaggtagg
      301 catatggcat ccacacacca atcatccaca gaaccctctt ccacaggtaa atctgaggaa
      361 acgaagaaag atgcttcgca agggagcggg caagactcca agaacgtaac cgttaccaaa
      421 ggtaccggtt cctccgccac ctcagctgcc attgtcaaga caggaggatc ccaaggcaaa
      481 gattcctcta ctacagcggg ctcttctagt actcagggac agaagttcag tactacacct
      541 accgacccga aaactttcag ctctgaccaa aaggagaaat ccaaaagccc agccaaagaa
      601 gtcccgtctg gtggcgatag taagtcccaa ggtgacacca agtctcaaag cgacgccaaa
      661 tcttctggac aaagtcaggg ccagtctaaa gacagcggca aatcatcttc cgacagtagc
      721 aagagtcact ctgtcatcgg agctgtcaaa gacgtcgttg caggcgccaa agatgtcgca
      781 ggaaaagccg tcgaggatgc tcctagcatc atgcatactg cagtcgatgc tgtgaagaac
      841 gcagccacga ctgtgaagga tgtggcatcg tcggctgcat cgactgtggc ggagaaggta
      901 gtcgatgctt accacagtgt ggtgggagac aagacggacg acaagaaaga gggcgagcac
      961 agcggcgaca agaaggacga ctccaaagct ggaagtggct ctggacaagg tggtgacaac
     1021 aagaagtctg aaggagagac ttctggccaa gcagaatcca gctctggcaa cgaaggagct
     1081 gctccagcca aaggccgtgg tcgtggacgg cctccagcag ctgctaaagg agttgctaag
     1141 ggtgctgcaa agggcgctgc cgcctccaaa ggagccaaga gcggtgctga atcctccaag
     1201 ggaggagaac agtcgtcagg agatatcgag atggcagatg cttcctccaa gggaggctcg
     1261 gaccagaggg attccgcggc gaccgttggc gaaggtggtg catcaggcag tgagggtgga
     1321 gctaagaaag gcagagggcg gggcgctggt aagaaagcgg atgcgggtga tacgtccgct
     1381 gagccgcctc ggcggtcgtc ccgcctgacg tcttcaggta caggggcggg ttccgctcca
     1441 gctgcagcga aaggcggagc gaagcgtgct gcttcttcct ccagtacacc ttccaacgct
     1501 aagaagcaag cgactggagg tgctggcaaa gctgctgcca ccaaagcaac tgctgccaaa
     1561 tcggcagcct ctaaagctcc ccagaatggc gcaggtgcca agaagaaggg aggaaaggct
     1621 ggaggacgga agaggaagta atctagatag gtaatctctg cttaaaagca cagaatctaa
     1681 gatccctgcc atttggcggg gattttttta tttgttttca ggaaataaat aatcgatcgc
     1741 gtaataaaat ctattattat ttttgtgaag aataaatttg ggtgcaatga gaatgcgcag
     1801 gccctttcgt ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc
     1861 ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc
     1921 gtcagcgggt gttggcgggt gtcggggctg gcttaactat gcggcatcag agcagattgt
     1981 actgagagtg caccataaaa ttgtaaacgt taatattttg ttaaaattcg cgttaaattt
     2041 ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc
     2101 aaaagaatag cccgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt
     2161 aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact
     2221 acgtgaacca tcacccaaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg
     2281 gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag
     2341 aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac
     2401 gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtactatgg
     2461 ttgctttgac gtatgcggtg tgaaataccg cacagatgcg taaggagaaa ataccgcatc
     2521 aggcgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     2581 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
     2641 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
     2701 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
     2761 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
     2821 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat
     2881 gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact
     2941 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
     3001 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
     3061 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
     3121 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
     3181 agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg
     3241 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
     3301 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
     3361 ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc
     3421 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
     3481 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
     3541 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
     3601 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     3661 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     3721 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     3781 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc
     3841 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     3901 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     3961 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     4021 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     4081 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     4141 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     4201 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     4261 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     4321 ggccttttgc tcacatagtc atgccccgcg cccaccggaa ggagctgact gggttgaagg
     4381 ctctcaaggg catcggtcga gatcccggtg cctaatgagt gagctaactt acattaattg
     4441 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     4501 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ccagggtggt ttttcttttc
     4561 accagtgaga cgggcaacag ctgattgccc ttcaccgcct ggccctgaga gagttgcagc
     4621 aagcggtcca cgctggtttg ccccagcagg cgaaaatcct gtttgatggt ggttaacggc
     4681 gggatataac atgagctgtc ttcggtatcg tcgtatccca ctaccgagat atccgcacca
     4741 acgcgcagcc cggactcggt aatggcgcgc attgcgccca gcgccatctg atcgttggca
     4801 accagcatcg cagtgggaac gatgccctca ttcagcattt gcatggtttg ttgaaaaccg
     4861 gacatggcac tccagtcgcc ttcccgttcc gctatcggct gaatttgatt gcgagtgaga
     4921 tatttatgcc agccagccag acgcagacgc gccgagacag aacttaatgg gcccgctaac
     4981 agcgcgattt gctggtgacc caatgcgacc agatgctcca cgcccagtcg cgtaccgtct
     5041 tcatgggaga aaataatact gttgatgggt gtctggtcag agacatcaag aaataacgcc
     5101 ggaacattag tgcaggcagc ttccacagca atggcatcct ggtcatccag cggatagtta
     5161 atgatcagcc cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg
     5221 acgccgcttc gttctaccat cgacaccacc acgctggcac ccagttgatc ggcgcgagat
     5281 ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca gactggaggt ggcaacgcca
     5341 atcagcaacg actgtttgcc cgccagttgt tgtgccacgc ggttgggaat gtaattcagc
     5401 tccgccatcg ccgcttccac tttttcccgc gttttcgcag aaacgtggct ggcctggttc
     5461 accacgcggg aaacggtctg ataagagaca ccggcatact ctgcgacatc gtataacgtt
     5521 actggtttca cattcaccac cctgaattga ctctcttccg ggcgctatca tgccataccg
     5581 cgaaaggttt tgcgccattc gatggtgtcc gggatctcga cgctctccct tatgcgactc
     5641 ctgcattagg aagcagccca gtagtaggtt gaggccgttg agcaccgccg ccgc
//
