LOCUS       sequence-372318-        4982 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of heat-resistant obscure (Hero) protein 
            Hero20.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4982)
  AUTHORS   Tsuboyama K, Osaki T, Matsuura-Suzuki E, Kozuka-Hata H, Okada Y, 
            Oyama M, Ikeuchi Y, Iwasaki S, Tomari Y
  TITLE     A widespread family of heat-resistant obscure (Hero) proteins 
            protect against protein instability and aggregation.
  JOURNAL   PLoS Biol. 2020 Mar 12;18(3):e3000632. doi: 
            10.1371/journal.pbio.3000632. eCollection 2020 Mar.
  PUBMED    32163402
REFERENCE   2  (bases 1 to 4982)
  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 
            Biol."; date: "2020-03-12"; volume: "18(3):e3000632. doi"; pages: " 
            10.1371/journal.pbio.3000632. eCollection 2020 Mar"
COMMENT     Sequence Label: pCold I-Hero20
FEATURES             Location/Qualifiers
     source          1..4982
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        17..83
                     /gene="cspA"
                     /label=cspA promoter
                     /note="promoter of the E. coli cold shock protein cspA gene
                     (Mitta et al., 1997)"
     protein_bind    86..102
                     /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           124..257
                     /gene="cspA"
                     /label=cspA 5'UTR
                     /note="5'UTR of the E. coli cold shock protein cspA gene 
                     (Mitta et al., 1997)"
     CDS             258..272
                     /codon_start=1
                     /gene="cspA"
                     /product="translation enhancing element for E. coli (Qing 
                     et al., 2004)"
                     /label=TEE
                     /translation="MNHKV"
     CDS             822..845
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             846..863
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             866..877
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     3'UTR           945..1089
                     /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(1130..1152)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     1252..1271
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(1288..1743)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1425..1446)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1637..1656
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        1843..1947
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1948..2016,2017..2808)
                     /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(2166..2185)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2979..3567
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3468..3487
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3739..4821)
                     /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     4783..4802
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4822..4899)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 gcaaggaatg gtgtggccga ttaatcataa atatgaaaaa taattgttgc atcacccgcc
       61 aatgcgtggc ttaatgcaca tcaaattgtg agcggataac aatttgatgt gctagcgcat
      121 atccagtgta gtaaggcaag tcccttcaag agttatcgtt gatacccctc gtagtgcaca
      181 ttcctttaac gcttcaaaat ctgtaaagca cgccatatcg ccgaaaggca cacttaatta
      241 ttaagaggta atacaccatg aatcacaaag tgatgagtgc tgccagagag tctcacccgc
      301 atggggtgaa gcgttcagcc tccccagacg acgatctggg atctagcaat tgggaggcag
      361 cagacttggg taatgaagag agaaaacaaa agttcttgag acttatgggt gcaggaaaga
      421 aagaacatac tggtcgtctt gttataggag atcacaaatc aacatctcac ttccgaaccg
      481 gggaagaaga caagaaaatt aatgaagaac tggagtctca atatcagcaa agtatggaca
      541 gtaaattatc aggaagatat cggcgacatt gtggacttgg cttcagtgag gtagaagacc
      601 atgatggaga aggtgatgtg gctggagatg atgatgatga cgatgatgat tcacctgatc
      661 ctgaaagtcc agatgattct gaaagcgatt cagagtcaga gaaagaagaa tctgctgaag
      721 aactccaagc tgctgagcac cctgatgaag tggaggatcc caaaaacaaa aaagatgcaa
      781 aaagcaatta taaaatgatg tttgttaaat ccagtggttc agactacaaa gacgatgacg
      841 acaagcatca tcatcatcat cattaatcga aggtaggcat atggagctcg gtaccctcga
      901 gggatccgaa ttcaagcttg tcgacctgca gtctagatag gtaatctctg cttaaaagca
      961 cagaatctaa gatccctgcc atttggcggg gattttttta tttgttttca ggaaataaat
     1021 aatcgatcgc gtaataaaat ctattattat ttttgtgaag aataaatttg ggtgcaatga
     1081 gaatgcgcag gccctttcgt ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca
     1141 tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc
     1201 gtcagggcgc gtcagcgggt gttggcgggt gtcggggctg gcttaactat gcggcatcag
     1261 agcagattgt actgagagtg caccataaaa ttgtaaacgt taatattttg ttaaaattcg
     1321 cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc
     1381 cttataaatc aaaagaatag cccgagatag ggttgagtgt tgttccagtt tggaacaaga
     1441 gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg
     1501 atggcccact acgtgaacca tcacccaaat caagtttttt ggggtcgagg tgccgtaaag
     1561 cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga
     1621 acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg
     1681 tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg
     1741 cgtactatgg ttgctttgac gtatgcggtg tgaaataccg cacagatgcg taaggagaaa
     1801 ataccgcatc aggcgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     1861 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
     1921 cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt
     1981 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta
     2041 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc
     2101 ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa
     2161 gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc
     2221 cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt
     2281 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact
     2341 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac
     2401 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata
     2461 ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta
     2521 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg
     2581 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat
     2641 aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt
     2701 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga
     2761 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa
     2821 gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
     2881 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     2941 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     3001 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     3061 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     3121 actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     3181 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     3241 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     3301 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     3361 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     3421 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     3481 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     3541 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     3601 gccttttgct ggccttttgc tcacatagtc atgccccgcg cccaccggaa ggagctgact
     3661 gggttgaagg ctctcaaggg catcggtcga gatcccggtg cctaatgagt gagctaactt
     3721 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     3781 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ccagggtggt
     3841 ttttcttttc accagtgaga cgggcaatag ctgattgccc ttcaccgcct ggccctgaga
     3901 gagttgcagc aagcggtcca cgctggtttg ccccagcagg cgaaaatcct gtttgatggt
     3961 ggttaacggc gggatataac atgagctgtc ttcggtatcg tcgtatccca ctaccgagat
     4021 atccgcacca acgcgcagcc cggactcggt aatggcgcgc attgcgccca gcgccatctg
     4081 atcgttggca accagcatcg cagtgggaac gatgccctca ttcagcattt gcatggtttg
     4141 ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc gctatcggct gaatttgatt
     4201 gcgagtgaga tatttatgcc agccagccag acgcagacgc gccgagacag aacttaatgg
     4261 gcccgctaac agcgcgattt gctggtgacc caatgcgacc agatgctcca cgcccagtcg
     4321 cgtaccgtct tcatgggaga aaataatact gttgatgggt gtctggtcag agacatcaag
     4381 aaataacgcc ggaacattag tgcaggcagc ttccacagca atggcatcct ggtcatccag
     4441 cggatagtta atgatcagcc cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt
     4501 acaggcttcg acgccgcttc gttctaccat cgacaccacc acgctggcac ccagttgatc
     4561 ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca gactggaggt
     4621 ggcaacgcca atcagcaacg actgtttgcc cgccagttgt tgtgccacgc ggttgggaat
     4681 gtaattcagc tccgccatcg ccgcttccac tttttcccgc gttttcgcag aaacgtggct
     4741 ggcctggttc accacgcggg aaacggtctg ataagagaca ccggcatact ctgcgacatc
     4801 gtataacgtt actggtttca cattcaccac cctgaattga ctctcttccg ggcgctatca
     4861 tgccataccg cgaaaggttt tgcgccattc gatggtgtcc gggatctcga cgctctccct
     4921 tatgcgactc ctgcattagg aagcagccca gtagtaggtt gaggccgttg agcaccgccg
     4981 cc
//
