LOCUS       sequence-403818-        6438 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pET-derived vector for expression of proteins N-terminally tagged 
            with 6xHis and maltose binding protein (multi-cloning sites: NdeI, 
            NotI, SpeI, SalI, PstI, SgfI, and XhoI).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6438)
  AUTHORS   Aoki K, Moriguchi H, Yoshioka T, Okawa K, Tabara H
  TITLE     In vitro analyses of the production and activity of secondary small 
            interfering RNAs in C. elegans.
  JOURNAL   EMBO J. 2007 Dec 12. 26(24):5007-19.
  PUBMED    18007599
REFERENCE   2  (bases 1 to 6438)
  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. 
            2007 Dec 12. 26(24):5007-19."
COMMENT     Sequence Label: pHIT198
FEATURES             Location/Qualifiers
     source          1..6438
                     /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"
     regulatory      1261..1270
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1303..1320
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1373..1391)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1387..1434
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1471..1926
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1558..1577)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1768..1789
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2018..2833)
                     /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(2018..2833)
                     /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     2741..2760
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2825..2844
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2955..3543
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3444..3463
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3697..3714
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3729..3868
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3794..3813)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3954..3976
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3970..4161)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4970..6052)
                     /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     6014..6033
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6053..6130)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6336..6355
                     /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 gccgccatgg atatcactag tcgactgcag cgatcgctcg agcaccacca ccaccaccac
     1321 tgagatccgg ctgctaacaa agcccgaaag gaagctgagt tggctgctgc caccgctgag
     1381 caataactag cataacccct tggggcctct aaacgggtct tgaggggttt tttgctgaaa
     1441 ggaggaacta tatccggatt ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg
     1501 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc
     1561 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
     1621 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
     1681 ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt
     1741 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca
     1801 accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt
     1861 taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgctta
     1921 caatttaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     1981 aatacattca aatatgtatc cgctcatgaa ttaattctta gaaaaactca tcgagcatca
     2041 aatgaaactg caatttattc atatcaggat tatcaatacc atatttttga aaaagccgtt
     2101 tctgtaatga aggagaaaac tcaccgaggc agttccatag gatggcaaga tcctggtatc
     2161 ggtctgcgat tccgactcgt ccaacatcaa tacaacctat taatttcccc tcgtcaaaaa
     2221 taaggttatc aagtgagaaa tcaccatgag tgacgactga atccggtgag aatggcaaaa
     2281 gtttatgcat ttctttccag acttgttcaa caggccagcc attacgctcg tcatcaaaat
     2341 cactcgcatc aaccaaaccg ttattcattc gtgattgcgc ctgagcgaga cgaaatacgc
     2401 gatcgctgtt aaaaggacaa ttacaaacag gaatcgaatg caaccggcgc aggaacactg
     2461 ccagcgcatc aacaatattt tcacctgaat caggatattc ttctaatacc tggaatgctg
     2521 ttttcccggg gatcgcagtg gtgagtaacc atgcatcatc aggagtacgg ataaaatgct
     2581 tgatggtcgg aagaggcata aattccgtca gccagtttag tctgaccatc tcatctgtaa
     2641 catcattggc aacgctacct ttgccatgtt tcagaaacaa ctctggcgca tcgggcttcc
     2701 catacaatcg atagattgtc gcacctgatt gcccgacatt atcgcgagcc catttatacc
     2761 catataaatc agcatccatg ttggaattta atcgcggcct agagcaagac gtttcccgtt
     2821 gaatatggct cataacaccc cttgtattac tgtttatgta agcagacagt tttattgttc
     2881 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     2941 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     3001 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     3061 aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag
     3121 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     3181 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     3241 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     3301 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     3361 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     3421 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     3481 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     3541 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
     3601 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga
     3661 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg
     3721 gaagagcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcaat
     3781 ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat acactccgct
     3841 atcgctacgt gactgggtca tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc
     3901 ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag
     3961 ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgaggcagc tgcggtaaag
     4021 ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc tgttcatccg cgtccagctc
     4081 gttgagtttc tccagaagcg ttaatgtctg gcttctgata aagcgggcca tgttaagggc
     4141 ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg ggatttctgt tcatgggggt
     4201 aatgataccg atgaaacgag agaggatgct cacgatacgg gttactgatg atgaacatgc
     4261 ccggttactg gaacgttgtg agggtaaaca actggcggta tggatgcggc gggaccagag
     4321 aaaaatcact cagggtcaat gccagcgctt cgttaataca gatgtaggtg ttccacaggg
     4381 tagccagcag catcctgcga tgcagatccg gaacataatg gtgcagggcg ctgacttccg
     4441 cgtttccaga ctttacgaaa cacggaaacc gaagaccatt catgttgttg ctcaggtcgc
     4501 agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt atcggtgatt cattctgcta
     4561 accagtaagg caaccccgcc agcctagccg ggtcctcaac gacaggagca cgatcatgcg
     4621 cacccgtggg gccgccatgc cggcgataat ggcctgcttc tcgccgaaac gtttggtggc
     4681 gggaccagtg acgaaggctt gagcgagggc gtgcaagatt ccgaataccg caagcgacag
     4741 gccgatcatc gtcgcgctcc agcgaaagcg gtcctcgccg aaaatgaccc agagcgctgc
     4801 cggcacctgt cctacgagtt gcatgataaa gaagacagtc ataagtgcgg cgacgatagt
     4861 catgccccgc gcccaccgga aggagctgac tgggttgaag gctctcaagg gcatcggtcg
     4921 agatcccggt gcctaatgag tgagctaact tacattaatt gcgttgcgct cactgcccgc
     4981 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     5041 aggcggtttg cgtattgggc gccagggtgg tttttctttt caccagtgag acgggcaaca
     5101 gctgattgcc cttcaccgcc tggccctgag agagttgcag caagcggtcc acgctggttt
     5161 gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa catgagctgt
     5221 cttcggtatc gtcgtatccc actaccgaga tatccgcacc aacgcgcagc ccggactcgg
     5281 taatggcgcg cattgcgccc agcgccatct gatcgttggc aaccagcatc gcagtgggaa
     5341 cgatgccctc attcagcatt tgcatggttt gttgaaaacc ggacatggca ctccagtcgc
     5401 cttcccgttc cgctatcggc tgaatttgat tgcgagtgag atatttatgc cagccagcca
     5461 gacgcagacg cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac
     5521 ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag aaaataatac
     5581 tgttgatggg tgtctggtca gagacatcaa gaaataacgc cggaacatta gtgcaggcag
     5641 cttccacagc aatggcatcc tggtcatcca gcggatagtt aatgatcagc ccactgacgc
     5701 gttgcgcgag aagattgtgc accgccgctt tacaggcttc gacgccgctt cgttctacca
     5761 tcgacaccac cacgctggca cccagttgat cggcgcgaga tttaatcgcc gcgacaattt
     5821 gcgacggcgc gtgcagggcc agactggagg tggcaacgcc aatcagcaac gactgtttgc
     5881 ccgccagttg ttgtgccacg cggttgggaa tgtaattcag ctccgccatc gccgcttcca
     5941 ctttttcccg cgttttcgca gaaacgtggc tggcctggtt caccacgcgg gaaacggtct
     6001 gataagagac accggcatac tctgcgacat cgtataacgt tactggtttc acattcacca
     6061 ccctgaattg actctcttcc gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt
     6121 cgatggtgtc cgggatctcg acgctctccc ttatgcgact cctgcattag gaagcagccc
     6181 agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg caaggagatg
     6241 gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa acaagcgctc
     6301 atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca
     6361 gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta gaggatcgag
     6421 atctcgatcc cgcgaaat
//
