LOCUS       sequence-403820-        7892 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of COH-3 C-terminal fragment with 6xHis and 
            maltose binding protein tags for use as an antigen.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7892)
  AUTHORS   Tabara H, Mitani S, Mochizuki M, Kohara Y, Nagata K
  TITLE     A small RNA system ensures accurate homologous pairing and unpaired 
            silencing of meiotic chromosomes.
  JOURNAL   EMBO J. 2023 Jun 1;42(11):e105002. doi: 10.15252/embj.2020105002. 
            Epub 2023 Apr 20.
  PUBMED    37078421
REFERENCE   2  (bases 1 to 7892)
  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: "EMBO J."; 
            date: "2023-06-1"; volume: "42(11):e105002. doi"; pages: " 
            10.15252/embj.2020105002. Epub 2023 Apr 20"
COMMENT     Sequence Label: pHIT334
FEATURES             Location/Qualifiers
     source          1..7892
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             100..117
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             127..144
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             151..1251
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="MKTEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
                     EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
                     IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
                     ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     primer_bind     1225..1248
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             2757..2774
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2827..2845)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2841..2888
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2925..3380
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3012..3031)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3222..3243
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3472..4287)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(3472..4287)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     4195..4214
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4279..4298
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4409..4997
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4898..4917
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5151..5168
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5183..5322
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5248..5267)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5408..5430
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5424..5615)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(6424..7506)
                     /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     7468..7487
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7507..7584)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7790..7809
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atataccatg ggcagcagcc atcatcatca tcatcacagc
      121 agcggcctgg tgccgcgcgg cagccataat atgaaaactg aagaaggtaa actggtaatc
      181 tggattaacg gcgataaagg ctataacggt ctcgctgaag tcggtaagaa attcgagaaa
      241 gataccggaa ttaaagtcac cgttgagcat ccggataaac tggaagagaa attcccacag
      301 gttgcggcaa ctggcgatgg ccctgacatt atcttctggg cacacgaccg ctttggtggc
      361 tacgctcaat ctggcctgtt ggctgaaatc accccggaca aagcgttcca ggacaagctg
      421 tatccgttta cctgggatgc cgtacgttac aacggcaagc tgattgctta cccgatcgct
      481 gttgaagcgt tatcgctgat ttataacaaa gatctgctgc cgaacccgcc aaaaacctgg
      541 gaagagatcc cggcgctgga taaagaactg aaagcgaaag gtaagagcgc gctgatgttc
      601 aacctgcaag aaccgtactt cacctggccg ctgattgctg ctgacggggg ttatgcgttc
      661 aagtatgaaa acggcaagta cgacattaaa gacgtgggcg tggataacgc tggcgcgaaa
      721 gcgggtctga ccttcctggt tgacctgatt aaaaacaaac acatgaatgc agacaccgat
      781 tactccatcg cagaagctgc ctttaataaa ggcgaaacag cgatgaccat caacggcccg
      841 tgggcatggt ccaacatcga caccagcaaa gtgaattatg gtgtaacggt actgccgacc
      901 ttcaagggtc aaccatccaa accgttcgtt ggcgtgctga gcgcaggtat taacgccgcc
      961 agtccgaaca aagagctggc aaaagagttc ctcgaaaact atctgctgac tgatgaaggt
     1021 ctggaagcgg ttaataaaga caaaccgctg ggtgccgtag cgctgaagtc ttacgaggaa
     1081 gagttggcga aagatccacg tattgccgcc actatggaaa acgcccagaa aggtgaaatc
     1141 atgccgaaca tcccgcagat gtccgctttc tggtatgccg tgcgtactgc ggtgatcaac
     1201 gccgccagcg gtcgtcagac tgtcgatgaa gccctgaaag acgcgcagac tcatatggcg
     1261 gccgcaggtg acaactctaa aacaccaaag aagaagagaa gaacgatggt tgaggaattt
     1321 cctctcactc ctaccccgat cagtgtggct ctaaaacaaa acattacaat gccagatgtt
     1381 ctcagtcttc acaagaacga ccaaattaac gttgaagacg atttggctgc accaacaatg
     1441 gaacaacttg aaatgatgta cgggatgttg gaatcagaaa atcggaacac agcgattgat
     1501 ttaacattca tcgatcgata ttcctccaac gattgccgtc aaaatgttca taatctgaat
     1561 ttggagactg attttcaacg cccatctgat gactttatcc ggagccagct catgacacct
     1621 accaaatcta ttccacaaga agaggaattt caaccagcca cacacctgaa ggaaatgctt
     1681 cttttccgcc aactccccat cacactagac gaaactgaga gaagaagaag catggaagaa
     1741 gaaatggagg aagcgaggaa ctctggaatc aacaccgagt ttattccgcc acaacaaatg
     1801 gatgttccac gacttgtctc ggaaaacatg gatctagatg acgaagtttc gattccaata
     1861 agaattccca aagttcctct tagtgctaca aagaaacaaa cgtctgagaa cgaaggagca
     1921 gaagaaggga tcaattataa aattccatta agagaaatgc atcacataat ggaagattat
     1981 tccagtcttc acttcaaaga cccggagcct ctataccgaa agacaagcaa agtgatgaat
     2041 ttaaaagagc tgttgaaccc aattccttac ttccttcgaa aaggatgcaa tacagacatg
     2101 tgggatctgt tcaaggtgaa aacgaggaag tttgattcca caattggcca cgatgtatct
     2161 gatactgaag aagaggacga agacgaggag actgagagaa gaaatcgact taaagctgaa
     2221 cttgagccct tggagaccat cgacttgtca cagatgaagt tcatgacgac tccactgaag
     2281 gataaaagtc ctatattgac tccagtgcat cgaccggaag aaattgagtt tcgtgaagag
     2341 ctcgaacttc cacggtttga acaatttgag ccgatgaatg tgaatccgat ttctccaaaa
     2401 atggaagagc cggcgctatt tgaagacaac tggaactcca aaccgacgtg gtcaaataca
     2461 agttctgaaa acattgcgga tgcgctcacc atccgtggga atattattcg ccagctggat
     2521 gataaaagcc ccttcgaggt cactctggat tcgatgatcc cggttgctgc gacatctcga
     2581 cgagaagctg ctcggacatt ctatacagtt cttgagctcc tcaaagaacg caagattaag
     2641 gccactcaac gtgctccgta tgaaaatatc gatttgctgc tatcaacgga tgatagtgaa
     2701 gatatcgacg atctggcaat ggctgatttc taagtcgact gcagcgatcg ctcgagcacc
     2761 accaccacca ccactgagat ccggctgcta acaaagcccg aaaggaagct gagttggctg
     2821 ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg
     2881 gttttttgct gaaaggagga actatatccg gattggcgaa tgggacgcgc cctgtagcgg
     2941 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     3001 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     3061 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     3121 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac
     3181 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     3241 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat
     3301 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa
     3361 aatattaacg cttacaattt aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     3421 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgaattaatt cttagaaaaa
     3481 ctcatcgagc atcaaatgaa actgcaattt attcatatca ggattatcaa taccatattt
     3541 ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg aggcagttcc ataggatggc
     3601 aagatcctgg tatcggtctg cgattccgac tcgtccaaca tcaatacaac ctattaattt
     3661 cccctcgtca aaaataaggt tatcaagtga gaaatcacca tgagtgacga ctgaatccgg
     3721 tgagaatggc aaaagtttat gcatttcttt ccagacttgt tcaacaggcc agccattacg
     3781 ctcgtcatca aaatcactcg catcaaccaa accgttattc attcgtgatt gcgcctgagc
     3841 gagacgaaat acgcgatcgc tgttaaaagg acaattacaa acaggaatcg aatgcaaccg
     3901 gcgcaggaac actgccagcg catcaacaat attttcacct gaatcaggat attcttctaa
     3961 tacctggaat gctgttttcc cggggatcgc agtggtgagt aaccatgcat catcaggagt
     4021 acggataaaa tgcttgatgg tcggaagagg cataaattcc gtcagccagt ttagtctgac
     4081 catctcatct gtaacatcat tggcaacgct acctttgcca tgtttcagaa acaactctgg
     4141 cgcatcgggc ttcccataca atcgatagat tgtcgcacct gattgcccga cattatcgcg
     4201 agcccattta tacccatata aatcagcatc catgttggaa tttaatcgcg gcctagagca
     4261 agacgtttcc cgttgaatat ggctcataac accccttgta ttactgttta tgtaagcaga
     4321 cagttttatt gttcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     4381 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     4441 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     4501 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc
     4561 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     4621 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     4681 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     4741 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     4801 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     4861 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     4921 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     4981 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     5041 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
     5101 ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag
     5161 tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta
     5221 tttcacaccg caatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca
     5281 gtatacactc cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca
     5341 cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg
     5401 accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg
     5461 cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca
     5521 tccgcgtcca gctcgttgag tttctccaga agcgttaatg tctggcttct gataaagcgg
     5581 gccatgttaa gggcggtttt ttcctgtttg gtcactgatg cctccgtgta agggggattt
     5641 ctgttcatgg gggtaatgat accgatgaaa cgagagagga tgctcacgat acgggttact
     5701 gatgatgaac atgcccggtt actggaacgt tgtgagggta aacaactggc ggtatggatg
     5761 cggcgggacc agagaaaaat cactcagggt caatgccagc gcttcgttaa tacagatgta
     5821 ggtgttccac agggtagcca gcagcatcct gcgatgcaga tccggaacat aatggtgcag
     5881 ggcgctgact tccgcgtttc cagactttac gaaacacgga aaccgaagac cattcatgtt
     5941 gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt
     6001 gattcattct gctaaccagt aaggcaaccc cgccagccta gccgggtcct caacgacagg
     6061 agcacgatca tgcgcacccg tggggccgcc atgccggcga taatggcctg cttctcgccg
     6121 aaacgtttgg tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa gattccgaat
     6181 accgcaagcg acaggccgat catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg
     6241 acccagagcg ctgccggcac ctgtcctacg agttgcatga taaagaagac agtcataagt
     6301 gcggcgacga tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt gaaggctctc
     6361 aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct aacttacatt aattgcgttg
     6421 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     6481 caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc ttttcaccag
     6541 tgagacgggc aacagctgat tgcccttcac cgcctggccc tgagagagtt gcagcaagcg
     6601 gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg atggtggtta acggcgggat
     6661 ataacatgag ctgtcttcgg tatcgtcgta tcccactacc gagatatccg caccaacgcg
     6721 cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag
     6781 catcgcagtg ggaacgatgc cctcattcag catttgcatg gtttgttgaa aaccggacat
     6841 ggcactccag tcgccttccc gttccgctat cggctgaatt tgattgcgag tgagatattt
     6901 atgccagcca gccagacgca gacgcgccga gacagaactt aatgggcccg ctaacagcgc
     6961 gatttgctgg tgacccaatg cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg
     7021 ggagaaaata atactgttga tgggtgtctg gtcagagaca tcaagaaata acgccggaac
     7081 attagtgcag gcagcttcca cagcaatggc atcctggtca tccagcggat agttaatgat
     7141 cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc
     7201 gcttcgttct accatcgaca ccaccacgct ggcacccagt tgatcggcgc gagatttaat
     7261 cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag
     7321 caacgactgt ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc
     7381 catcgccgct tccacttttt cccgcgtttt cgcagaaacg tggctggcct ggttcaccac
     7441 gcgggaaacg gtctgataag agacaccggc atactctgcg acatcgtata acgttactgg
     7501 tttcacattc accaccctga attgactctc ttccgggcgc tatcatgcca taccgcgaaa
     7561 ggttttgcgc cattcgatgg tgtccgggat ctcgacgctc tcccttatgc gactcctgca
     7621 ttaggaagca gcccagtagt aggttgaggc cgttgagcac cgccgccgca aggaatggtg
     7681 catgcaagga gatggcgccc aacagtcccc cggccacggg gcctgccacc atacccacgc
     7741 cgaaacaagc gctcatgagc ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg
     7801 cgatataggc gccagcaacc gcacctgtgg cgccggtgat gccggccacg atgcgtccgg
     7861 cgtagaggat cgagatctcg atcccgcgaa at
//
