LOCUS       sequence-490612-        6587 bp    DNA     circular SYN 24-OCT-2025
DEFINITION  For production of recombinant siRNAs (pro-siRNAs) in E. coli..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6587)
  AUTHORS   Kaur G, Cheung HC, Xu W, Wong JV, Chan FF, Li Y, McReynolds L, Huang
            L
  TITLE     Milligram scale production of potent recombinant small interfering 
            RNAs in Escherichia coli.
  JOURNAL   Biotechnol Bioeng. 2018 Jun 5. doi: 10.1002/bit.26740.
  PUBMED    29873060
REFERENCE   2  (bases 1 to 6587)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 24, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Biotechnol 
            Bioeng. 2018 Jun 5. doi: 10.1002/bit.26740."
COMMENT     Sequence Label: pET-pro-siRNA-V2
FEATURES             Location/Qualifiers
     source          1..6587
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        15..33
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    34..58
                     /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             73..95
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             106..123
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     promoter        1326..1344
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(1437..1455)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1462..1479
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1532..1550)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1546..1593
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1630..2085
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1717..1736)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1927..1948
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2177..2992)
                     /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(2177..2992)
                     /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     2900..2919
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2984..3003
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3114..3702
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3603..3622
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3856..3873
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3888..4027
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3953..3972)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4113..4135
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4129..4320)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5129..6211)
                     /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     6173..6192
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6212..6289)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6495..6514
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 cgatcccgcg aaattaatac gactcactat aggggaattg tgagcggata acaattcccc
       61 tctagaaata attttgttta actttaagaa ggagatatac atatgcatca tcaccaccat
      121 cacgaacgtg caatccaggg caacgacgcc cgtgaacagg ccaacagcga acgctgggat
      181 ggtggcagcg gcggtacaac cagccctttt aaactgccgg acgaaagccc gagttggacc
      241 gaatggcgcc tgcataacga tgagaccaac agcaaccagg acaacccgct gggttttaaa
      301 gaaagctggg gcttcggcaa agtggtgttc aagcgctatc tgcgctacga tcgtaccgaa
      361 gccagcctgc atcgcgttct gggtagctgg accggtgaca gcgtgaatta tgccgccagc
      421 cgttttttcg gcttcgatca gatcggctgc acctacagca ttcgcttccg cggtgtgagc
      481 attaccgtta gcggcggtag ccgtacactg cagcacctgt gtgaaatggc cattcgcagt
      541 aagcaagagc tgctgcagct ggccccgatt gaggtggaaa gtaatgtgag ccgtggctgt
      601 ccggagggca cccaaacctt cgaaaaagag agtgagtaat gatgaacccc atcgtaatta
      661 atcggcttca acggaagctg ggctacactt ttaatcatca ggaactgttg cagcaggcat
      721 taactcatcg tagtgccagc agtaaacata acgagcgttt agaattttta ggcgactcta
      781 ttctgagcta cgttatcgcc aatgcgcttt atcaccgttt ccctcgtgtg gatgaaggcg
      841 atatgagccg gatgcgcgcc acgctggtcc gtggcaatac gctggcggaa ctggcgcgcg
      901 aatttgagtt aggcgagtgc ttacgtttag ggccaggtga acttaaaagc ggtggatttc
      961 gtcgtgagtc aattctcgcc gacaccgtcg aagcattaat tggtggcgta ttcctcgaca
     1021 gtgatattca aaccgtcgag aaattaatcc tcaactggta tcaaactcgt ttggacgaaa
     1081 ttagcccagg cgataaacaa aaagatccga aaacgcgctt gcaagaatat ttgcagggtc
     1141 gccatctgcc gctgccgact tatctggtag tccaggtacg tggcgaagcg cacgatcagg
     1201 aatttactat ccactgccag gtcagcggcc tgagtgaacc ggtggttggc acaggttcaa
     1261 gccgtcgtaa ggctgagcag gctgccgccg aacaggcgtt gaaaaaactg gagctggaat
     1321 gaatctaata cgactcacta taggtgagct cgatctcgag ttgactattg cttaacgata
     1381 tacgtactcg tttaaatctc tgatcatcga atctaaaagt cgacactgcg gccgccccta
     1441 tagtgagtcg tattagtcga gcaccaccac caccaccact gagatccggc tgctaacaaa
     1501 gcccgaaagg aagctgagtt ggctgctgcc accgctgagc aataactagc ataacccctt
     1561 ggggcctcta aacgggtctt gaggggtttt ttgctgaaag gaggaactat atccggattg
     1621 gcgaatggga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
     1681 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct
     1741 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
     1801 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac
     1861 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct
     1921 ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt
     1981 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac
     2041 aaaaatttaa cgcgaatttt aacaaaatat taacgcttac aatttaggtg gcacttttcg
     2101 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     2161 gctcatgaat taattcttag aaaaactcat cgagcatcaa atgaaactgc aatttattca
     2221 tatcaggatt atcaatacca tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact
     2281 caccgaggca gttccatagg atggcaagat cctggtatcg gtctgcgatt ccgactcgtc
     2341 caacatcaat acaacctatt aatttcccct cgtcaaaaat aaggttatca agtgagaaat
     2401 caccatgagt gacgactgaa tccggtgaga atggcaaaag tttatgcatt tctttccaga
     2461 cttgttcaac aggccagcca ttacgctcgt catcaaaatc actcgcatca accaaaccgt
     2521 tattcattcg tgattgcgcc tgagcgagac gaaatacgcg atcgctgtta aaaggacaat
     2581 tacaaacagg aatcgaatgc aaccggcgca ggaacactgc cagcgcatca acaatatttt
     2641 cacctgaatc aggatattct tctaatacct ggaatgctgt tttcccgggg atcgcagtgg
     2701 tgagtaacca tgcatcatca ggagtacgga taaaatgctt gatggtcgga agaggcataa
     2761 attccgtcag ccagtttagt ctgaccatct catctgtaac atcattggca acgctacctt
     2821 tgccatgttt cagaaacaac tctggcgcat cgggcttccc atacaatcga tagattgtcg
     2881 cacctgattg cccgacatta tcgcgagccc atttataccc atataaatca gcatccatgt
     2941 tggaatttaa tcgcggccta gagcaagacg tttcccgttg aatatggctc ataacacccc
     3001 ttgtattact gtttatgtaa gcagacagtt ttattgttca tgaccaaaat cccttaacgt
     3061 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     3121 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     3181 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     3241 gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac
     3301 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     3361 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     3421 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     3481 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     3541 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     3601 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     3661 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     3721 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
     3781 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
     3841 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat
     3901 tttctcctta cgcatctgtg cggtatttca caccgcaatg gtgcactctc agtacaatct
     3961 gctctgatgc cgcatagtta agccagtata cactccgcta tcgctacgtg actgggtcat
     4021 ggctgcgccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc
     4081 ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc
     4141 accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag
     4201 cgattcacag atgtctgcct gttcatccgc gtccagctcg ttgagtttct ccagaagcgt
     4261 taatgtctgg cttctgataa agcgggccat gttaagggcg gttttttcct gtttggtcac
     4321 tgatgcctcc gtgtaagggg gatttctgtt catgggggta atgataccga tgaaacgaga
     4381 gaggatgctc acgatacggg ttactgatga tgaacatgcc cggttactgg aacgttgtga
     4441 gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga aaaatcactc agggtcaatg
     4501 ccagcgcttc gttaatacag atgtaggtgt tccacagggt agccagcagc atcctgcgat
     4561 gcagatccgg aacataatgg tgcagggcgc tgacttccgc gtttccagac tttacgaaac
     4621 acggaaaccg aagaccattc atgttgttgc tcaggtcgca gacgttttgc agcagcagtc
     4681 gcttcacgtt cgctcgcgta tcggtgattc attctgctaa ccagtaaggc aaccccgcca
     4741 gcctagccgg gtcctcaacg acaggagcac gatcatgcgc acccgtgggg ccgccatgcc
     4801 ggcgataatg gcctgcttct cgccgaaacg tttggtggcg ggaccagtga cgaaggcttg
     4861 agcgagggcg tgcaagattc cgaataccgc aagcgacagg ccgatcatcg tcgcgctcca
     4921 gcgaaagcgg tcctcgccga aaatgaccca gagcgctgcc ggcacctgtc ctacgagttg
     4981 catgataaag aagacagtca taagtgcggc gacgatagtc atgccccgcg cccaccggaa
     5041 ggagctgact gggttgaagg ctctcaaggg catcggtcga gatcccggtg cctaatgagt
     5101 gagctaactt acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     5161 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     5221 ccagggtggt ttttcttttc accagtgaga cgggcaacag ctgattgccc ttcaccgcct
     5281 ggccctgaga gagttgcagc aagcggtcca cgctggtttg ccccagcagg cgaaaatcct
     5341 gtttgatggt ggttaacggc gggatataac atgagctgtc ttcggtatcg tcgtatccca
     5401 ctaccgagat gtccgcacca acgcgcagcc cggactcggt aatggcgcgc attgcgccca
     5461 gcgccatctg atcgttggca accagcatcg cagtgggaac gatgccctca ttcagcattt
     5521 gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc gctatcggct
     5581 gaatttgatt gcgagtgaga tatttatgcc agccagccag acgcagacgc gccgagacag
     5641 aacttaatgg gcccgctaac agcgcgattt gctggtgacc caatgcgacc agatgctcca
     5701 cgcccagtcg cgtaccgtct tcatgggaga aaataatact gttgatgggt gtctggtcag
     5761 agacatcaag aaataacgcc ggaacattag tgcaggcagc ttccacagca atggcatcct
     5821 ggtcatccag cggatagtta atgatcagcc cactgacgcg ttgcgcgaga agattgtgca
     5881 ccgccgcttt acaggcttcg acgccgcttc gttctaccat cgacaccacc acgctggcac
     5941 ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca
     6001 gactggaggt ggcaacgcca atcagcaacg actgtttgcc cgccagttgt tgtgccacgc
     6061 ggttgggaat gtaattcagc tccgccatcg ccgcttccac tttttcccgc gttttcgcag
     6121 aaacgtggct ggcctggttc accacgcggg aaacggtctg ataagagaca ccggcatact
     6181 ctgcgacatc gtataacgtt actggtttca cattcaccac cctgaattga ctctcttccg
     6241 ggcgctatca tgccataccg cgaaaggttt tgcgccattc gatggtgtcc gggatctcga
     6301 cgctctccct tatgcgactc ctgcattagg aagcagccca gtagtaggtt gaggccgttg
     6361 agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag tcccccggcc
     6421 acggggcctg ccaccatacc cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc
     6481 cgatcttccc catcggtgat gtcggcgata taggcgccag caaccgcacc tgtggcgccg
     6541 gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga tcgatct
//
