LOCUS       sequence-403810-        7238 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of CEC-4 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 7238)
  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 7238)
  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: pHIT517
FEATURES             Location/Qualifiers
     source          1..7238
                     /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"
     protein_bind    1981..1992
                     /label=ZF binding site
                     /bound_moiety="CCR5TC zinc-finger domain"
                     /note="target sequence for the CCR5TC zinc-finger domain 
                     from the CCR5-224 (+) zinc-finger nuclease (Perez et al., 
                     2008; Gross et al., 2013)"
     CDS             2103..2120
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2173..2191)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2187..2234
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2271..2726
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2358..2377)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2568..2589
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2818..3633)
                     /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(2818..3633)
                     /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     3541..3560
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3625..3644
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3755..4343
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4244..4263
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4497..4514
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4529..4668
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4594..4613)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4754..4776
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4770..4961)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5770..6852)
                     /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     6814..6833
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6853..6930)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7136..7155
                     /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 cgaagaagac tgttgaagga gaacacggta ctccgaaaac taatttcaca
     1321 aagaaagaaa cgagtaaaaa tcacgatgat ttcaaaaaaa ttattggaca taaagtggtt
     1381 gaagagcatt atgtggaata tgaagtagaa ctgaccagcg gaaaaactat tacagcaact
     1441 gagtttgact ttaagggtga tgattctctt ctgtcaacct acaaaaagaa ggtaaccaag
     1501 caatctgatg atagcagcgg agaatatgcc gtggagcgtg tgttggctca tcgaaaggtg
     1561 aaaggcagcc cactttatct agttcaatgg aaaggatacc cgcatccagt gtggaactcg
     1621 gaaatgtggg aggaagatct tgataactgc aaggatttgc tagctgctta caagaagcat
     1681 caagaggatc tcaaaatcgc tcaaactccg aagaagactc caagcaagac accaaagaaa
     1741 actccaaaat cgttgaaaag aagagctctc actccatccg atgacgaaga agaggcggga
     1801 ccgattgcgc ctgagcctaa aaagacacca aaacaatcta caaagaagct gaagcgtacc
     1861 acctccccgg aaacaaactt agttgaaaag agcaagaaga aagcaattcc ggatttggaa
     1921 aaccatacct tagatcaaga aaagaacgat gtcatcgaac gtgtcgaaga aattcaagag
     1981 gatgaggatg acgacgacga gcaaagagaa gaagtggtga caaccgcccc agtggagaca
     2041 aagtcaagat ggggattcgg ttcatggaaa tggttttaag tcgactgcag cgatcgctcg
     2101 agcaccacca ccaccaccac tgagatccgg ctgctaacaa agcccgaaag gaagctgagt
     2161 tggctgctgc caccgctgag caataactag cataacccct tggggcctct aaacgggtct
     2221 tgaggggttt tttgctgaaa ggaggaacta tatccggatt ggcgaatggg acgcgccctg
     2281 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
     2341 cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
     2401 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
     2461 gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg
     2521 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt
     2581 ccaaactgga acaacactca accctatctc ggtctattct tttgatttat aagggatttt
     2641 gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt
     2701 taacaaaata ttaacgctta caatttaggt ggcacttttc ggggaaatgt gcgcggaacc
     2761 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgaa ttaattctta
     2821 gaaaaactca tcgagcatca aatgaaactg caatttattc atatcaggat tatcaatacc
     2881 atatttttga aaaagccgtt tctgtaatga aggagaaaac tcaccgaggc agttccatag
     2941 gatggcaaga tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa tacaacctat
     3001 taatttcccc tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag tgacgactga
     3061 atccggtgag aatggcaaaa gtttatgcat ttctttccag acttgttcaa caggccagcc
     3121 attacgctcg tcatcaaaat cactcgcatc aaccaaaccg ttattcattc gtgattgcgc
     3181 ctgagcgaga cgaaatacgc gatcgctgtt aaaaggacaa ttacaaacag gaatcgaatg
     3241 caaccggcgc aggaacactg ccagcgcatc aacaatattt tcacctgaat caggatattc
     3301 ttctaatacc tggaatgctg ttttcccggg gatcgcagtg gtgagtaacc atgcatcatc
     3361 aggagtacgg ataaaatgct tgatggtcgg aagaggcata aattccgtca gccagtttag
     3421 tctgaccatc tcatctgtaa catcattggc aacgctacct ttgccatgtt tcagaaacaa
     3481 ctctggcgca tcgggcttcc catacaatcg atagattgtc gcacctgatt gcccgacatt
     3541 atcgcgagcc catttatacc catataaatc agcatccatg ttggaattta atcgcggcct
     3601 agagcaagac gtttcccgtt gaatatggct cataacaccc cttgtattac tgtttatgta
     3661 agcagacagt tttattgttc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     3721 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     3781 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     3841 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     3901 tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     3961 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     4021 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     4081 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     4141 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     4201 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     4261 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     4321 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     4381 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
     4441 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg
     4501 agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt acgcatctgt
     4561 gcggtatttc acaccgcaat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
     4621 aagccagtat acactccgct atcgctacgt gactgggtca tggctgcgcc ccgacacccg
     4681 ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa
     4741 gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc
     4801 gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc
     4861 tgttcatccg cgtccagctc gttgagtttc tccagaagcg ttaatgtctg gcttctgata
     4921 aagcgggcca tgttaagggc ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg
     4981 ggatttctgt tcatgggggt aatgataccg atgaaacgag agaggatgct cacgatacgg
     5041 gttactgatg atgaacatgc ccggttactg gaacgttgtg agggtaaaca actggcggta
     5101 tggatgcggc gggaccagag aaaaatcact cagggtcaat gccagcgctt cgttaataca
     5161 gatgtaggtg ttccacaggg tagccagcag catcctgcga tgcagatccg gaacataatg
     5221 gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa cacggaaacc gaagaccatt
     5281 catgttgttg ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt
     5341 atcggtgatt cattctgcta accagtaagg caaccccgcc agcctagccg ggtcctcaac
     5401 gacaggagca cgatcatgcg cacccgtggg gccgccatgc cggcgataat ggcctgcttc
     5461 tcgccgaaac gtttggtggc gggaccagtg acgaaggctt gagcgagggc gtgcaagatt
     5521 ccgaataccg caagcgacag gccgatcatc gtcgcgctcc agcgaaagcg gtcctcgccg
     5581 aaaatgaccc agagcgctgc cggcacctgt cctacgagtt gcatgataaa gaagacagtc
     5641 ataagtgcgg cgacgatagt catgccccgc gcccaccgga aggagctgac tgggttgaag
     5701 gctctcaagg gcatcggtcg agatcccggt gcctaatgag tgagctaact tacattaatt
     5761 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
     5821 atcggccaac gcgcggggag aggcggtttg cgtattgggc gccagggtgg tttttctttt
     5881 caccagtgag acgggcaaca gctgattgcc cttcaccgcc tggccctgag agagttgcag
     5941 caagcggtcc acgctggttt gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg
     6001 cgggatataa catgagctgt cttcggtatc gtcgtatccc actaccgaga tatccgcacc
     6061 aacgcgcagc ccggactcgg taatggcgcg cattgcgccc agcgccatct gatcgttggc
     6121 aaccagcatc gcagtgggaa cgatgccctc attcagcatt tgcatggttt gttgaaaacc
     6181 ggacatggca ctccagtcgc cttcccgttc cgctatcggc tgaatttgat tgcgagtgag
     6241 atatttatgc cagccagcca gacgcagacg cgccgagaca gaacttaatg ggcccgctaa
     6301 cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc
     6361 ttcatgggag aaaataatac tgttgatggg tgtctggtca gagacatcaa gaaataacgc
     6421 cggaacatta gtgcaggcag cttccacagc aatggcatcc tggtcatcca gcggatagtt
     6481 aatgatcagc ccactgacgc gttgcgcgag aagattgtgc accgccgctt tacaggcttc
     6541 gacgccgctt cgttctacca tcgacaccac cacgctggca cccagttgat cggcgcgaga
     6601 tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc agactggagg tggcaacgcc
     6661 aatcagcaac gactgtttgc ccgccagttg ttgtgccacg cggttgggaa tgtaattcag
     6721 ctccgccatc gccgcttcca ctttttcccg cgttttcgca gaaacgtggc tggcctggtt
     6781 caccacgcgg gaaacggtct gataagagac accggcatac tctgcgacat cgtataacgt
     6841 tactggtttc acattcacca ccctgaattg actctcttcc gggcgctatc atgccatacc
     6901 gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg acgctctccc ttatgcgact
     6961 cctgcattag gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga
     7021 atggtgcatg caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac
     7081 ccacgccgaa acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga
     7141 tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc
     7201 gtccggcgta gaggatcgag atctcgatcc cgcgaaat
//
