LOCUS       sequence-416918-        8724 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  protein expression in ecoli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8724)
  AUTHORS   Fitzgerald ME, Vela A, Pyle AM
  TITLE     Dicer-related helicase 3 forms an obligate dimer for recognizing 
            22G-RNA.
  JOURNAL   Nucleic Acids Res. 2014 Apr;42(6):3919-30. doi: 10.1093/nar/gkt1383.
            Epub 2014 Jan 15.
  PUBMED    24435798
REFERENCE   2  (bases 1 to 8724)
  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"; doi: 
            "10.1093/nar/gkt1383"; journalName: "Nucleic Acids Res"; date: 
            "2014-04"; volume: "42"; issue: "6"; pages: "3919-30"
COMMENT     Sequence Label: pET28sumo-MDA5-Full-length
FEATURES             Location/Qualifiers
     source          1..8724
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             63..80
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(133..151)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      147..194
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      231..686
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(318..337)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     528..549
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(778..1593)
                     /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(778..1593)
                     /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     1501..1520
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1585..1604
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1715..2303
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2204..2223
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2457..2474
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2489..2628
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2554..2573)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2714..2736
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2730..2921)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3730..4812)
                     /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     4774..4793
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4813..4890)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5096..5115
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5199..5218
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5199..5217
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5218..5242
                     /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             5257..5279
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5299..5316
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             5326..5343
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             5350..5643
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
ORIGIN
        1 agacaagctt aggtatttat tcggcgcaaa gtgcgtcggg tgatgctgcc aacttagtcg
       61 agcaccacca ccaccaccac tgagatccgg ctgctaacaa agcccgaaag gaagctgagt
      121 tggctgctgc caccgctgag caataactag cataacccct tggggcctct aaacgggtct
      181 tgaggggttt tttgctgaaa ggaggaacta tatccggatt ggcgaatggg acgcgccctg
      241 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
      301 cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
      361 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
      421 gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg
      481 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt
      541 ccaaactgga acaacactca accctatctc ggtctattct tttgatttat aagggatttt
      601 gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt
      661 taacaaaata ttaacgctta caatttaggt ggcacttttc ggggaaatgt gcgcggaacc
      721 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgaa ttaattctta
      781 gaaaaactca tcgagcatca aatgaaactg caatttattc atatcaggat tatcaatacc
      841 atatttttga aaaagccgtt tctgtaatga aggagaaaac tcaccgaggc agttccatag
      901 gatggcaaga tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa tacaacctat
      961 taatttcccc tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag tgacgactga
     1021 atccggtgag aatggcaaaa gtttatgcat ttctttccag acttgttcaa caggccagcc
     1081 attacgctcg tcatcaaaat cactcgcatc aaccaaaccg ttattcattc gtgattgcgc
     1141 ctgagcgaga cgaaatacgc gatcgctgtt aaaaggacaa ttacaaacag gaatcgaatg
     1201 caaccggcgc aggaacactg ccagcgcatc aacaatattt tcacctgaat caggatattc
     1261 ttctaatacc tggaatgctg ttttcccggg gatcgcagtg gtgagtaacc atgcatcatc
     1321 aggagtacgg ataaaatgct tgatggtcgg aagaggcata aattccgtca gccagtttag
     1381 tctgaccatc tcatctgtaa catcattggc aacgctacct ttgccatgtt tcagaaacaa
     1441 ctctggcgca tcgggcttcc catacaatcg atagattgtc gcacctgatt gcccgacatt
     1501 atcgcgagcc catttatacc catataaatc agcatccatg ttggaattta atcgcggcct
     1561 agagcaagac gtttcccgtt gaatatggct cataacaccc cttgtattac tgtttatgta
     1621 agcagacagt tttattgttc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     1681 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     1741 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     1801 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     1861 tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     1921 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     1981 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     2041 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     2101 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     2161 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     2221 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     2281 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     2341 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
     2401 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg
     2461 agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt acgcatctgt
     2521 gcggtatttc acaccgcaat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
     2581 aagccagtat acactccgct atcgctacgt gactgggtca tggctgcgcc ccgacacccg
     2641 ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa
     2701 gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc
     2761 gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc
     2821 tgttcatccg cgtccagctc gttgagtttc tccagaagcg ttaatgtctg gcttctgata
     2881 aagcgggcca tgttaagggc ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg
     2941 ggatttctgt tcatgggggt aatgataccg atgaaacgag agaggatgct cacgatacgg
     3001 gttactgatg atgaacatgc ccggttactg gaacgttgtg agggtaaaca actggcggta
     3061 tggatgcggc gggaccagag aaaaatcact cagggtcaat gccagcgctt cgttaataca
     3121 gatgtaggtg ttccacaggg tagccagcag catcctgcga tgcagatccg gaacataatg
     3181 gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa cacggaaacc gaagaccatt
     3241 catgttgttg ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt
     3301 atcggtgatt cattctgcta accagtaagg caaccccgcc agcctagccg ggtcctcaac
     3361 gacaggagca cgatcatgcg cacccgtggg gccgccatgc cggcgataat ggcctgcttc
     3421 tcgccgaaac gtttggtggc gggaccagtg acgaaggctt gagcgagggc gtgcaagatt
     3481 ccgaataccg caagcgacag gccgatcatc gtcgcgctcc agcgaaagcg gtcctcgccg
     3541 aaaatgaccc agagcgctgc cggcacctgt cctacgagtt gcatgataaa gaagacagtc
     3601 ataagtgcgg cgacgatagt catgccccgc gcccaccgga aggagctgac tgggttgaag
     3661 gctctcaagg gcatcggtcg agatcccggt gcctaatgag tgagctaact tacattaatt
     3721 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
     3781 atcggccaac gcgcggggag aggcggtttg cgtattgggc gccagggtgg tttttctttt
     3841 caccagtgag acgggcaaca gctgattgcc cttcaccgcc tggccctgag agagttgcag
     3901 caagcggtcc acgctggttt gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg
     3961 cgggatataa catgagctgt cttcggtatc gtcgtatccc actaccgaga tatccgcacc
     4021 aacgcgcagc ccggactcgg taatggcgcg cattgcgccc agcgccatct gatcgttggc
     4081 aaccagcatc gcagtgggaa cgatgccctc attcagcatt tgcatggttt gttgaaaacc
     4141 ggacatggca ctccagtcgc cttcccgttc cgctatcggc tgaatttgat tgcgagtgag
     4201 atatttatgc cagccagcca gacgcagacg cgccgagaca gaacttaatg ggcccgctaa
     4261 cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc
     4321 ttcatgggag aaaataatac tgttgatggg tgtctggtca gagacatcaa gaaataacgc
     4381 cggaacatta gtgcaggcag cttccacagc aatggcatcc tggtcatcca gcggatagtt
     4441 aatgatcagc ccactgacgc gttgcgcgag aagattgtgc accgccgctt tacaggcttc
     4501 gacgccgctt cgttctacca tcgacaccac cacgctggca cccagttgat cggcgcgaga
     4561 tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc agactggagg tggcaacgcc
     4621 aatcagcaac gactgtttgc ccgccagttg ttgtgccacg cggttgggaa tgtaattcag
     4681 ctccgccatc gccgcttcca ctttttcccg cgttttcgca gaaacgtggc tggcctggtt
     4741 caccacgcgg gaaacggtct gataagagac accggcatac tctgcgacat cgtataacgt
     4801 tactggtttc acattcacca ccctgaattg actctcttcc gggcgctatc atgccatacc
     4861 gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg acgctctccc ttatgcgact
     4921 cctgcattag gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga
     4981 atggtgcatg caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac
     5041 ccacgccgaa acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga
     5101 tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc
     5161 gtccggcgta gaggatcgag atctcgatcc cgcgaaatta atacgactca ctatagggga
     5221 attgtgagcg gataacaatt cccctctaga aataattttg tttaacttta agaaggagat
     5281 atacatatgg gcagcagcca tcatcatcat catcacggca gcggcctggt gccgcgcggc
     5341 agcgctagca tgtcggactc agaagtcaat caagaagcta agccagaggt caagccagaa
     5401 gtcaagcctg agactcacat caatttaaag gtgtccgatg gatcttcaga gatcttcttc
     5461 aagatcaaaa agaccactcc tttaagaagg ctgatggaag cgttcgctaa aagacagggt
     5521 aaggaaatgg actccttaag attcttgtac gacggtatta gaattcaagc tgatcagacc
     5581 cctgaagatt tggacatgga ggataacgat attattgagg ctcacagaga acagattggt
     5641 ggttccatgt cgaatgggta ttccacagac gagaatttcc gctatctcat ctcgtgcttc
     5701 agggccaggg tgaaaatgta catccaggtg gagcctgtgc tggactacct gacctttctg
     5761 cctgcagagg tgaaggagca gattcagagg acagtcgcca cctccgggaa catgcaggca
     5821 gttgaactgc tgctgagcac cttggagaag ggagtctggc accttggttg gactcgggaa
     5881 ttcgtggagg ccctccggag aaccggcagc cctctggccg cccgctacat gaaccctgag
     5941 ctcacggact tgccctctcc atcgtttgag aacgctcatg atgaatatct ccaactgctg
     6001 aacctccttc agcccactct ggtggacaag cttctagtta gagacgtctt ggataagtgc
     6061 atggaggagg aactgttgac aattgaagac agaaaccgga ttgctgctgc agaaaacaat
     6121 ggaaatgaat caggtgtaag agagctacta aaaaggattg tgcagaaaga aaactggttc
     6181 tctgcatttc tgaatgttct tcgtcaaaca ggaaacaatg aacttgtcca agagttaaca
     6241 ggctctgatt gctcagaaag caatgcagag attgagaatt tatcacaagt tgatggtcct
     6301 caagtggaag agcaacttct ttcaaccaca gttcagccaa atctggagaa ggaggtctgg
     6361 ggcatggaga ataactcatc agaatcatct tttgcagatt cttctgtagt ttcagaatca
     6421 gacacaagtt tggcagaagg aagtgtcagc tgcttagatg aaagtcttgg acataacagc
     6481 aacatgggca gtgattcagg caccatggga agtgattcag atgaagagaa tgtggcagca
     6541 agagcatccc cggagccaga actccagctc aggccttacc aaatggaagt tgcccagcca
     6601 gccttggaag ggaagaatat catcatctgc ctccctacag ggagtggaaa aaccagagtg
     6661 gctgtttaca ttgccaagga tcacttagac aagaagaaaa aagcatctga gcctggaaaa
     6721 gttatagttc ttgtcaataa ggtactgcta gttgaacagc tcttccgcaa ggagttccaa
     6781 ccatttttga agaaatggta tcgtgttatt ggattaagtg gtgataccca actgaaaata
     6841 tcatttccag aagttgtcaa gtcctgtgat attattatca gtacagctca aatccttgaa
     6901 aactccctct taaacttgga aaatggagaa gatgctggtg ttcaattgtc agacttttcc
     6961 ctcattatca ttgatgaatg tcatcacacc aacaaagaag cagtgtataa taacatcatg
     7021 aggcattatt tgatgcagaa gttgaaaaac aatagactca agaaagaaaa caaaccagtg
     7081 attccccttc ctcagatact gggactaaca gcttcacctg gtgttggagg ggccacgaag
     7141 caagccaaag ctgaagaaca cattttaaaa ctatgtgcca atcttgatgc atttactatt
     7201 aaaactgtta aagaaaacct tgatcaactg aaaaaccaaa tacaggagcc atgcaagaag
     7261 tttgccattg cagatgcaac cagagaagat ccatttaaag agaaacttct agaaataatg
     7321 acaaggattc aaacttattg tcaaatgagt ccaatgtcag attttggaac tcaaccctat
     7381 gaacaatggg ccattcaaat ggaaaaaaaa gctgcaaaag aaggaaatcg caaagaacgt
     7441 gtttgtgcag aacatttgag gaagtacaat gaggccctac aaattaatga cacaattcga
     7501 atgatagatg cgtatactca tcttgaaact ttctataatg aagagaaaga taagaagttt
     7561 gcagtcatag aagatgatag tgatgagggt ggtgatgatg agtattgtga tggtgatgaa
     7621 gatgaggatg atttaaagaa acctttgaaa ctggatgaaa cagatagatt tctcatgact
     7681 ttattttttg aaaacaataa aatgttgaaa aggctggctg aaaacccaga atatgaaaat
     7741 gaaaagctga ccaaattaag aaataccata atggagcaat atactaggac tgaggaatca
     7801 gcacgaggaa taatctttac aaaaacacga cagagtgcat atgcgctttc ccagtggatt
     7861 actgaaaatg aaaaatttgc tgaagtagga gtcaaagccc accatctgat tggagctgga
     7921 cacagcagtg agttcaaacc catgacacag aatgaacaaa aagaagtcat tagtaaattt
     7981 cgcactggaa aaataaatct gcttatcgct accacagtgg cagaagaagg tctggatatt
     8041 aaagaatgta acattgttat ccgttatggt ctcgtcacca atgaaatagc catggtccag
     8101 gcccgtggtc gagccagagc tgatgagagc acctacgtcc tggttgctca cagtggttca
     8161 ggagttatcg aacatgagac agttaatgat ttccgagaga agatgatgta taaagctata
     8221 cattgtgttc aaaatatgaa accagaggag tatgctcata agattttgga attacagatg
     8281 caaagtataa tggaaaagaa aatgaaaacc aagagaaata ttgccaagca ttacaagaat
     8341 aacccatcac taataacttt cctttgcaaa aactgcagtg tgctagcctg ttctggggaa
     8401 gatatccatg taattgagaa aatgcatcac gtcaatatga ccccagaatt caaggaactt
     8461 tacattgtaa gagaaaacaa agcactgcaa aagaagtgtg ccgactatca aataaatggt
     8521 gaaatcatct gcaaatgtgg ccaggcttgg ggaacaatga tggtgcacaa aggcttagat
     8581 ttgccttgtc tcaaaataag gaattttgta gtggttttca aaaataattc aacaaagaaa
     8641 caatacaaaa agtgggtaga attacctatc acatttccca atcttgacta ttcagaatgc
     8701 tgtttattta gtgatgagga ttag
//
