LOCUS       sequence-403749-        7115 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of CEC-5 N-terminal fragment with 6xHis and 
            maltose binding protein tags for in vitro assays.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7115)
  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 7115)
  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: pHIT521
FEATURES             Location/Qualifiers
     source          1..7115
                     /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             1980..1997
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2050..2068)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2064..2111
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2148..2603
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2235..2254)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2445..2466
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2695..3510)
                     /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(2695..3510)
                     /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     3418..3437
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3502..3521
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3632..4220
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4121..4140
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4374..4391
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4406..4545
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4471..4490)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4631..4653
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4647..4838)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5647..6729)
                     /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     6691..6710
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6730..6807)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7013..7032
                     /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 gccgctatgg caaagaaatc atccggtagt gcaccatctc gtgcttctgg aagatcagga
     1321 aagcaaacca ctcacttcca ggttgtgcac gatgccacga aacaccgggc aaagcgtagc
     1381 aaatcgtcta aatctggaga ctcgaagagc tcttctcagg acgcttcagt caagaagatt
     1441 cttgatcaca aactattcaa cacgcactac gttgaatata atgtcctcct ttcaaatggc
     1501 aggaaaatga tggccactga gttcgatttt gagggcaaaa aggagcagaa actattggcc
     1561 gattacaagt ataagatcac caaccaagat gatgatccag atgaggattt tgctgtcgag
     1621 aaaatcattg ctcaccgctt cagcggaaag aagaacaagc cactgttttt ggtcatgtgg
     1681 cgtggatacc caaacccggt gtctcactcg gagatgtggg agaatgagtt gtcgaactgc
     1741 aaagatcttc ttgaagccta caaagattct catgacatga aaccaccacc ggtcaagaag
     1801 tctaacaaga agaagggaag caagaaatcg ccgaaaaagt ccgctaaatc gaaggatgat
     1861 gactcttccg acgaagagga agaagaggaa gtcaaagaga aatctccaaa gaaatcctcg
     1921 aaatccacaa agagagctgc ttctgatgat gattaagtcg actgcagcga tcgctcgagc
     1981 accaccacca ccaccactga gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg
     2041 ctgctgccac cgctgagcaa taactagcat aaccccttgg ggcctctaaa cgggtcttga
     2101 ggggtttttt gctgaaagga ggaactatat ccggattggc gaatgggacg cgccctgtag
     2161 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
     2221 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
     2281 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     2341 cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata
     2401 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
     2461 aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc
     2521 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa
     2581 caaaatatta acgcttacaa tttaggtggc acttttcggg gaaatgtgcg cggaacccct
     2641 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgaatta attcttagaa
     2701 aaactcatcg agcatcaaat gaaactgcaa tttattcata tcaggattat caataccata
     2761 tttttgaaaa agccgtttct gtaatgaagg agaaaactca ccgaggcagt tccataggat
     2821 ggcaagatcc tggtatcggt ctgcgattcc gactcgtcca acatcaatac aacctattaa
     2881 tttcccctcg tcaaaaataa ggttatcaag tgagaaatca ccatgagtga cgactgaatc
     2941 cggtgagaat ggcaaaagtt tatgcatttc tttccagact tgttcaacag gccagccatt
     3001 acgctcgtca tcaaaatcac tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg
     3061 agcgagacga aatacgcgat cgctgttaaa aggacaatta caaacaggaa tcgaatgcaa
     3121 ccggcgcagg aacactgcca gcgcatcaac aatattttca cctgaatcag gatattcttc
     3181 taatacctgg aatgctgttt tcccggggat cgcagtggtg agtaaccatg catcatcagg
     3241 agtacggata aaatgcttga tggtcggaag aggcataaat tccgtcagcc agtttagtct
     3301 gaccatctca tctgtaacat cattggcaac gctacctttg ccatgtttca gaaacaactc
     3361 tggcgcatcg ggcttcccat acaatcgata gattgtcgca cctgattgcc cgacattatc
     3421 gcgagcccat ttatacccat ataaatcagc atccatgttg gaatttaatc gcggcctaga
     3481 gcaagacgtt tcccgttgaa tatggctcat aacacccctt gtattactgt ttatgtaagc
     3541 agacagtttt attgttcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     3601 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     3661 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     3721 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc
     3781 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     3841 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     3901 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     3961 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     4021 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     4081 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     4141 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     4201 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     4261 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     4321 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     4381 cagtgagcga ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg
     4441 gtatttcaca ccgcaatggt gcactctcag tacaatctgc tctgatgccg catagttaag
     4501 ccagtataca ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg acacccgcca
     4561 acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct
     4621 gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcrtcacc gaaacgcgcg
     4681 aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt
     4741 tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta atgtctggct tctgataaag
     4801 cgggccatgt taagggcggt tttttcctgt ttggtcactg atgcctccgt gtaaggggga
     4861 tttctgttca tgggggtaat gataccgatg aaacgagaga ggatgctcac gatacgggtt
     4921 actgatgatg aacatgcccg gttactggaa cgttgtgagg gtaaacaact ggcggtatgg
     4981 atgcggcggg accagagaaa aatcactcag ggtcaatgcc agcgcttcgt taatacagat
     5041 gtaggtgttc cacagggtag ccagcagcat cctgcgatgc agatccggaa cataatggtg
     5101 cagggcgctg acttccgcgt ttccagactt tacgaaacac ggaaaccgaa gaccattcat
     5161 gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc
     5221 ggtgattcat tctgctaacc agtaaggcaa ccccgccagc ctagccgggt cctcaacgac
     5281 aggagcacga tcatgcgcac ccgtggggcc gccatgccgg cgataatggc ctgcttctcg
     5341 ccgaaacgtt tggtggcggg accagtgacg aaggcttgag cgagggcgtg caagattccg
     5401 aataccgcaa gcgacaggcc gatcatcgtc gcgctccagc gaaagcggtc ctcgccgaaa
     5461 atgacccaga gcgctgccgg cacctgtcct acgagttgca tgataaagaa gacagtcata
     5521 agtgcggcga cgatagtcat gccccgcgcc caccggaagg agctgactgg gttgaaggct
     5581 ctcaagggca tcggtcgaga tcccggtgcc taatgagtga gctaacttac attaattgcg
     5641 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     5701 ggccaacgcg cggggagagg cggtttgcgt attgggcgcc agggtggttt ttcttttcac
     5761 cagtgagacg ggcaacagct gattgccctt caccgcctgg ccctgagaga gttgcagcaa
     5821 gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg
     5881 gatataacat gagctgtctt cggtatcgtc gtatcccact accgagatat ccgcaccaac
     5941 gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc gccatctgat cgttggcaac
     6001 cagcatcgca gtgggaacga tgccctcatt cagcatttgc atggtttgtt gaaaaccgga
     6061 catggcactc cagtcgcctt cccgttccgc tatcggctga atttgattgc gagtgagata
     6121 tttatgccag ccagccagac gcagacgcgc cgagacagaa cttaatgggc ccgctaacag
     6181 cgcgatttgc tggtgaccca atgcgaccag atgctccacg cccagtcgcg taccgtcttc
     6241 atgggagaaa ataatactgt tgatgggtgt ctggtcagag acatcaagaa ataacgccgg
     6301 aacattagtg caggcagctt ccacagcaat ggcatcctgg tcatccagcg gatagttaat
     6361 gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac aggcttcgac
     6421 gccgcttcgt tctaccatcg acaccaccac gctggcaccc agttgatcgg cgcgagattt
     6481 aatcgccgcg acaatttgcg acggcgcgtg cagggccaga ctggaggtgg caacgccaat
     6541 cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt aattcagctc
     6601 cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa acgtggctgg cctggttcac
     6661 cacgcgggaa acggtctgat aagagacacc ggcatactct gcgacatcgt ataacgttac
     6721 tggtttcaca ttcaccaccc tgaattgact ctcttccggg cgctatcatg ccataccgcg
     6781 aaaggttttg cgccattcga tggtgtccgg gatctcgacg ctctccctta tgcgactcct
     6841 gcattaggaa gcagcccagt agtaggttga ggccgttgag caccgccgcc gcaaggaatg
     6901 gtgcatgcaa ggagatggcg cccaacagtc ccccggccac ggggcctgcc accataccca
     6961 cgccgaaaca agcgctcatg agcccgaagt ggcgagcccg atcttcccca tcggtgatgt
     7021 cggcgatata ggcgccagca accgcacctg tggcgccggt gatgccggcc acgatgcgtc
     7081 cggcgtagag gatcgagatc tcgatcccgc gaaat
//
