LOCUS       sequence-403742-        7853 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of HIM-1 internal 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 7853)
  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 7853)
  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: pHIT320
FEATURES             Location/Qualifiers
     source          1..7853
                     /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             2718..2735
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2788..2806)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2802..2849
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2886..3341
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2973..2992)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3183..3204
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3433..4248)
                     /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(3433..4248)
                     /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     4156..4175
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4240..4259
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4370..4958
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4859..4878
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5112..5129
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5144..5283
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5209..5228)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5369..5391
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5385..5576)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(6385..7467)
                     /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     7429..7448
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7468..7545)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7751..7770
                     /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 gccgcagaga agccaagcag tttgcgtgtt agaaagtatg cggatttgat tcatggtgca
     1321 ccaattaaca agccagttgc taagaaatgt cgtgtcacta tgaactacaa atattctgac
     1381 ggaaaagtca aggcattcac cagaggcgtc aataatggaa catctgaaca tcttcttgat
     1441 ggtcaaactg tcacaagtgc agcatattca caagaaatgg aaagtatcaa tatcttcatc
     1501 aaggcaagaa actttctggt atatcaggga gctatcgaaa atattgccat gaaaactcca
     1561 aaagaacgta ctcaactctt cgaggagctc tcacgatcac acgagtttca agcagagtat
     1621 gaaagattga aagttgagat gacaaaagct gaagatgata ctcaacacaa tatgaacaag
     1681 agacgaggaa tcgctcaaga gaaacgtgag gcaaaaatgg agaaagatga agctgaaaag
     1741 tatcaaacca tgaagaatga gctggctgca aaatcgacga tgctatttct ccatcaattg
     1801 ttccattgcg aacggacgat cgacgaatca aaagaggaga tcaacgctca gaaaaagacg
     1861 attgcaagtc tcgaagcaac taggtcgaaa gaagaagcga aaatcgctgc ggttcatcag
     1921 gagcaccgga aagctctccg cgaggttcag aagatgacaa gaaaactcga tcagaaagag
     1981 acggatttgg ctgaaaagca acaaaatatg ctgacattga aagtttccgt tgctcacgag
     2041 cacaagaagc tggaaattgc gaagaagatg cttgccgctg cagaaagtaa ggcagagaac
     2101 aactcgactc agctggcgga tttgaagaaa agcaaaaaag agctagaaaa gaagaaggcc
     2161 gcatatgaag cagagattca ggatatgatg caaagaggcg agttaaatct ttcggatgaa
     2221 caagtacgtg agtacggaca gctgaaagat caagctcagc gagaatcagc aatggttcaa
     2281 agagagctat tgatggccga acaagtgttc gaaggtgata agagttctct aaatcatgag
     2341 cttcgacgcc aaaaggagca tcaagaaaga gttaaagcga aggaaggtga tgtgcgaaga
     2401 attgaaactc aaattgcaac tcttgctcaa agaatcaagg aaactgaaga agagacaaag
     2461 atcttgaaag ctgatcttaa gaagattgaa aatgacgttg ttattgataa atcagcagca
     2521 gcggaataca acaaagagct tgtagctgtc gttcgtcaac tctccgaagc ttctggagat
     2581 tctgcagaag gagaaagaaa tcaaagaaga acggaggcac ttgaaggatt gaaaaagaat
     2641 ttcccggaga gtgtgtatgg tagacttgtc gatctctgtc aaccgtctca ttaagtcgac
     2701 tgcagcgatc gctcgagcac caccaccacc accactgaga tccggctgct aacaaagccc
     2761 gaaaggaagc tgagttggct gctgccaccg ctgagcaata actagcataa ccccttgggg
     2821 cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg aactatatcc ggattggcga
     2881 atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
     2941 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
     3001 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
     3061 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag
     3121 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa
     3181 tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga
     3241 tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa
     3301 atttaacgcg aattttaaca aaatattaac gcttacaatt taggtggcac ttttcgggga
     3361 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     3421 atgaattaat tcttagaaaa actcatcgag catcaaatga aactgcaatt tattcatatc
     3481 aggattatca ataccatatt tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc
     3541 gaggcagttc cataggatgg caagatcctg gtatcggtct gcgattccga ctcgtccaac
     3601 atcaatacaa cctattaatt tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc
     3661 atgagtgacg actgaatccg gtgagaatgg caaaagttta tgcatttctt tccagacttg
     3721 ttcaacaggc cagccattac gctcgtcatc aaaatcactc gcatcaacca aaccgttatt
     3781 cattcgtgat tgcgcctgag cgagacgaaa tacgcgatcg ctgttaaaag gacaattaca
     3841 aacaggaatc gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa tattttcacc
     3901 tgaatcagga tattcttcta atacctggaa tgctgttttc ccggggatcg cagtggtgag
     3961 taaccatgca tcatcaggag tacggataaa atgcttgatg gtcggaagag gcataaattc
     4021 cgtcagccag tttagtctga ccatctcatc tgtaacatca ttggcaacgc tacctttgcc
     4081 atgtttcaga aacaactctg gcgcatcggg cttcccatac aatcgataga ttgtcgcacc
     4141 tgattgcccg acattatcgc gagcccattt atacccatat aaatcagcat ccatgttgga
     4201 atttaatcgc ggcctagagc aagacgtttc ccgttgaata tggctcataa caccccttgt
     4261 attactgttt atgtaagcag acagttttat tgttcatgac caaaatccct taacgtgagt
     4321 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     4381 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     4441 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     4501 agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     4561 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     4621 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     4681 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     4741 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     4801 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     4861 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     4921 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     4981 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg
     5041 attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa
     5101 cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc
     5161 tccttacgca tctgtgcggt atttcacacc gcaatggtgc actctcagta caatctgctc
     5221 tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg ggtcatggct
     5281 gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca
     5341 tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg
     5401 tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat
     5461 tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag aagcgttaat
     5521 gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt ggtcactgat
     5581 gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa acgagagagg
     5641 atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg ttgtgagggt
     5701 aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg tcaatgccag
     5761 cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc tgcgatgcag
     5821 atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta cgaaacacgg
     5881 aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt
     5941 cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc ccgccagcct
     6001 agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc catgccggcg
     6061 ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg
     6121 agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc gctccagcga
     6181 aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac gagttgcatg
     6241 ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca ccggaaggag
     6301 ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta atgagtgagc
     6361 taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     6421 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag
     6481 ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca ccgcctggcc
     6541 ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt
     6601 gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt atcccactac
     6661 cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc
     6721 catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca gcatttgcat
     6781 ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta tcggctgaat
     6841 ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg agacagaact
     6901 taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat gctccacgcc
     6961 cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct ggtcagagac
     7021 atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg catcctggtc
     7081 atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc
     7141 cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc tggcacccag
     7201 ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca gggccagact
     7261 ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt
     7321 gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac
     7381 gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg catactctgc
     7441 gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct cttccgggcg
     7501 ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga tctcgacgct
     7561 ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg ccgttgagca
     7621 ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc ccggccacgg
     7681 ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat
     7741 cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg gcgccggtga
     7801 tgccggccac gatgcgtccg gcgtagagga tcgagatctc gatcccgcga aat
//
