LOCUS       sequence-372322-        5594 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of heat-resistant obscure (Hero) protein 
            Hero45.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5594)
  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 5594)
  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-Hero45
FEATURES             Location/Qualifiers
     source          1..5594
                     /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             1434..1457
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1458..1475
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1478..1489
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     3'UTR           1557..1701
                     /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(1742..1764)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     1864..1883
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(1900..2355)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2037..2058)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2249..2268
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2455..2559
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2560..2628,2629..3420)
                     /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(2778..2797)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3591..4179
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4080..4099
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(4351..5433)
                     /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     5395..5414
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5434..5511)
                     /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 tgatgcctgg ccatctccaa gaaggctttg
      301 gctgcgttgt taccaatcgc tttgatcagc tgtttgacga tgaaagtgat ccgttcgaag
      361 tgctcaaagc tgcggagaac aaaaaaaagg aggcaggagg cggtggtgtt gggggccctg
      421 gtgccaaatc tgcagcacag gcagctgcac agacgaacag caatgcggcg ggtaaacagc
      481 tgcgcaaaga atcccagaaa gatcgcaaaa atcccttacc cccgagtgtc ggtgtggtgg
      541 acaagaagga ggaaacccaa ccgccggtag cgttgaaaaa agagggcatt cgtcgcgttg
      601 ggcgacgccc ggatcaacag ctgcaaggcg agggtaaaat cattgatcgc cgaccagaac
      661 ggcgtccacc tcgcgaacgc cgttttgaga aaccgttgga agaaaaaggc gaaggtgggg
      721 aattcagcgt agatcgcccg atcattgacc gtcccatacg tggccgaggt ggcttaggcc
      781 gtggtcgtgg cggtcgggga cgtggcatgg gtcgcgggga tggcttcgac tcccggggga
      841 aacgcgaatt cgatcgccat tcgggatcag accgcagcgg cctgaaacac gaagacaaac
      901 gtgggggttc aggctcgcac aattggggca ctgtgaagga tgagcttacc gatctggatc
      961 agtccaatgt gaccgaggaa acgccggaag gcgaagaaca tcatccggtg gctgataccg
     1021 agaacaaaga aaacgaagtt gaagaggtga aggaagaagg cccgaaggag atgacactgg
     1081 acgaatggaa agcgatccaa aacaaagacc gtgcgaaagt cgagttcaac attcggaaac
     1141 cgaacgaagg tgcggatggt cagtggaaaa agggatttgt ccttcacaag agcaaaagcg
     1201 aggaagcgca tgccgaagac agcgtcatgg atcatcactt ccgtaaacca gccaatgaca
     1261 ttacgtcaca gctggaaatc aactttggcg atctaggtcg tcctggccgt ggagggagag
     1321 ggggccgtgg tggtaggggt cgcggtggcc gcccgaatcg tggatctcgc actgacaaat
     1381 cttcggccag tgccccggat gtagatgatc cagaagcgtt tccagctctg gccgactaca
     1441 aagacgatga cgacaagcat catcatcatc atcattaatc gaaggtaggc atatggagct
     1501 cggtaccctc gagggatccg aattcaagct tgtcgacctg cagtctagat aggtaatctc
     1561 tgcttaaaag cacagaatct aagatccctg ccatttggcg gggatttttt tatttgtttt
     1621 caggaaataa ataatcgatc gcgtaataaa atctattatt atttttgtga agaataaatt
     1681 tgggtgcaat gagaatgcgc aggccctttc gtctcgcgcg tttcggtgat gacggtgaaa
     1741 acctctgaca catgcagctc ccggagacgg tcacagcttg tctgtaagcg gatgccggga
     1801 gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc tggcttaact
     1861 atgcggcatc agagcagatt gtactgagag tgcaccataa aattgtaaac gttaatattt
     1921 tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa
     1981 tcggcaaaat cccttataaa tcaaaagaat agcccgagat agggttgagt gttgttccag
     2041 tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg
     2101 tctatcaggg cgatggccca ctacgtgaac catcacccaa atcaagtttt ttggggtcga
     2161 ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga gcttgacggg
     2221 gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg
     2281 cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgcg cttaatgcgc
     2341 cgctacaggg cgcgtactat ggttgctttg acgtatgcgg tgtgaaatac cgcacagatg
     2401 cgtaaggaga aaataccgca tcaggcgtca ggtggcactt ttcggggaaa tgtgcgcgga
     2461 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     2521 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
     2581 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
     2641 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
     2701 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg
     2761 agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag
     2821 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
     2881 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
     2941 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc
     3001 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg
     3061 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg
     3121 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
     3181 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
     3241 tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg
     3301 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
     3361 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa
     3421 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
     3481 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     3541 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     3601 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     3661 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     3721 cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct
     3781 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     3841 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     3901 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     3961 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     4021 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     4081 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     4141 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     4201 ttacggttcc tggccttttg ctggcctttt gctcacatag tcatgccccg cgcccaccgg
     4261 aaggagctga ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg tgcctaatga
     4321 gtgagctaac ttacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     4381 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     4441 cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc ccttcaccgc
     4501 ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc
     4561 ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat cgtcgtatcc
     4621 cactaccgag atatccgcac caacgcgcag cccggactcg gtaatggcgc gcattgcgcc
     4681 cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct cattcagcat
     4741 ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt ccgctatcgg
     4801 ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac gcgccgagac
     4861 agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga ccagatgctc
     4921 cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg gtgtctggtc
     4981 agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag caatggcatc
     5041 ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga gaagattgtg
     5101 caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca ccacgctggc
     5161 acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc
     5221 cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt gttgtgccac
     5281 gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc gcgttttcgc
     5341 agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga caccggcata
     5401 ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt gactctcttc
     5461 cgggcgctat catgccatac cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc
     5521 gacgctctcc cttatgcgac tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt
     5581 tgagcaccgc cgcc
//
