LOCUS       sequence-389058-        6525 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  siRNA vector expressing TMV P19 and a 720-long EGFP sequence as 
            inverted repeat.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6525)
  AUTHORS   Villa R, Renzi S, Dotti S, Lucchini F
  TITLE     siRNAs pools generated in Escherichia coli exhibit strong 
            RNA-interference activity against influenza virus genomic sequences.
  JOURNAL   Virology. 2022 Dec 31;579:38-45. doi: 10.1016/j.virol.2022.12.013.
  PUBMED    36599198
REFERENCE   2  (bases 1 to 6525)
  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: "Virology.";
            date: "2022-12-31"; volume: "579:38-45. doi"; pages: " 
            10.1016/j.virol.2022.12.013"
COMMENT     Sequence Label: pBAD-P19-EGFP
FEATURES             Location/Qualifiers
     source          1..6525
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             522..539
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     546..565
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        546..564
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             join(582..584,585..587,588..1301)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(582..584,585..587,588..1298)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     1235..1256
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             complement(join(1488..2000,2001..2003,2004..2006))
                     /codon_start=1
                     /product="N-terminal fragment of mVenus for use in 
                     bimolecular fluorescence complementation (BiFC) (Kodama and
                     Hu, 2010)"
                     /label=VN173
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIE"
     primer_bind     complement(2234..2252)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2320..2424
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2425..2493,2494..3285)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2643..2662)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3327..3782
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3414..3433)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3624..3645
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     rep_origin      3893..4481
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4382..4401
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4635..4652
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4667..4807
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4733..4752)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5053..5072
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     CDS             complement(5252..6130)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     promoter        6157..6441
                     /gene="araBAD"
                     /label=araBAD promoter
                     /note="promoter of the L-arabinose operon of E. coli; the 
                     araC regulatory gene is transcribed in the opposite 
                     direction (Guzman et al., 1995)"
     primer_bind     6364..6383
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     RBS             6468..6490
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             6507..6524
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
ORIGIN
        1 gatccatgga acgagctata caaggaaacg acgctaggga acaagctaac agtgaacgtt
       61 gggatggagg atcaggaggt accacttctc ccttcaaact tcctgacgaa agtccgagtt
      121 ggactgagtg gcggctacat aacgatgaga cgaattcgaa tcaagataat ccccttggtt
      181 tcaaggaaag ctggggtttc gggaaagttg tatttaagag atatctcaga tacgacagga
      241 cggaagcttc actgcacaga gtccttggat cttggacggg agattcggtt aactatgcag
      301 catctcgatt tttcggtttc gaccagatcg gatgtaccta tagtattcgg tttcgaggag
      361 ttagtatcac cgtttctgga gggtcgcgaa ctcttcagca tctctgtgag atggcaattc
      421 ggtctaagca agaactgcta cagcttgccc caatcgaagt ggaaagtaat gtatcaagag
      481 gatgccctga aggtactcaa accttcgaaa aagaaagcga gcaccaccac caccaccact
      541 gaatctaata cgactcacta taggggagct actagtagct catggtgagc aagggcgagg
      601 agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca
      661 agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt
      721 tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgacct
      781 acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt
      841 ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact
      901 acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga
      961 agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca
     1021 acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag gtgaacttca
     1081 agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca
     1141 cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagtccg
     1201 ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg
     1261 ccgccgggat cactctcggc atggacgagc tgtacaagta gctcgagtct agagggcccg
     1321 tttaaacccg ctgatcagcc tcgagggacc atgtgatcgc gcttctcgtt ggggtctttg
     1381 ctcagggcgg actgggtgct caggtagtgg ttgtcgggca gcagcacggg gccgtcgccg
     1441 atgggggtgt tctgctggta gtggtcggcg agctgcacgc tgccgtcctc gatgttgtgg
     1501 cggatcttga agttcacctt gatgccgttc ttctgcttgt cggccatgat atagacgttg
     1561 tggctgttgt agttgtactc cagcttgtgc cccaggatgt tgccgtcctc cttgaagtcg
     1621 atgcccttca gctcgatgcg gttcaccagg gtgtcgccct cgaacttcac ctcggcgcgg
     1681 gtcttgtagt tgccgtcgtc cttgaagaag atggtgcgct cctggacgta gccttcgggc
     1741 atggcggact tgaagaagtc gtgctgcttc atgtggtcgg ggtagcggct gaagcactgc
     1801 acgccgtagg tcagggtggt cacgagggtg ggccagggca cgggcagctt gccggtggtg
     1861 cagatgaact tcagggtcag cttgccgtag gtggcatcgc cctcgccctc gccggacacg
     1921 ctgaacttgt ggccgtttac gtcgccgtcc agctcgacca ggatgggcac caccccggtg
     1981 aacagctcct cgcccttgct caccatgcgg ccgcatcgtg actgactgac gatctgcctc
     2041 gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc agctcccgga gacggtcaca
     2101 gcttgtctgt aagcggatgc cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt
     2161 ggcgggtgtc ggggcgcagc catgacccag tcacgtagcg atagcggagt gtataattct
     2221 tgaagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg
     2281 gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
     2341 tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt
     2401 caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc
     2461 ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa
     2521 gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt
     2581 aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt
     2641 ctgctatgtg gcgcggtatt atcccgtgtt gacgccgggc aagagcaact cggtcgccgc
     2701 atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg
     2761 gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg
     2821 gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac
     2881 atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca
     2941 aacgacgagc gtgacaccac gatgcctgca gcaatggcaa caacgttgcg caaactatta
     3001 actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat
     3061 aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa
     3121 tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag
     3181 ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat
     3241 agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt
     3301 tactcatata tactttagat tgatttacgc gccctgtagc ggcgcattaa gcgcggcggg
     3361 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
     3421 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     3481 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     3541 tttgggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
     3601 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc
     3661 tatctcgggc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa
     3721 aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgtttacaat
     3781 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg
     3841 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     3901 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     3961 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     4021 cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact
     4081 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     4141 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     4201 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     4261 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     4321 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     4381 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     4441 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     4501 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc
     4561 ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc
     4621 gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg atgcggtatt
     4681 ttctccttac gcatctgtgc ggtatttcac accgcatatg gtgcactctc agtacaatct
     4741 gctctgatgc cgcatagtta agccagtata cactccgcta tcgctacgtg actgggtcat
     4801 ggctgcgccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc
     4861 ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc
     4921 accgtcatca ccgaaacgcg cgaggcagaa ggagatggcg cccaacagtc ccccggccac
     4981 ggggcctgcc accataccca cgccgaaaca agcgctcatg agcccgaagt ggcgagcccg
     5041 atcttcccca tcggtgatgt cggcgatata ggcgccagca accgcacctg tggcgccggt
     5101 gatgccggcc acgatgcgtc cggcgtagag gatctgctca tgtttgacag cttatcatcg
     5161 atgcataatg tgcctgtcaa atggacgaag cagggattct gcaaacccta tgctactccg
     5221 tcaagccgtc aattgtctga ttcgttacca attatgacaa cttgacggct acatcattca
     5281 ctttttcttc acaaccggca cggaactcgc tcgggctggc cccggtgcat tttttaaata
     5341 cccgcgagaa atagagttga tcgtcaaaac caacattgcg accgacggtg gcgataggca
     5401 tccgggtggt gctcaaaagc agcttcgcct ggctgatacg ttggtcctcg cgccagctta
     5461 agacgctaat ccctaactgc tggcggaaaa gatgtgacag acgcgacggc gacaagcaaa
     5521 catgctgtgc gacgctggcg atatcaaaat tgctgtctgc caggtgatcg ctgatgtact
     5581 gacaagcctc gcgtacccga ttatccatcg gtggatggag cgactcgtta atcgcttcca
     5641 tgcgccgcag taacaattgc tcaagcagat ttatcgccag cagctccgaa tagcgccctt
     5701 ccccttgccc ggcgttaatg atttgcccaa acaggtcgct gaaatgcggc tggtgcgctt
     5761 catccgggcg aaagaacccc gtattggcaa atattgacgg ccagttaagc cattcatgcc
     5821 agtaggcgcg cggacgaaag taaacccact ggtgatacca ttcgcgagcc tccggatgac
     5881 gaccgtagtg atgaatctct cctggcggga acagcaaaat atcacccggt cggcaaacaa
     5941 attctcgtcc ctgatttttc accaccccct gaccgcgaat ggtgagattg agaatataac
     6001 ctttcattcc cagcggtcgg tcgataaaaa aatcgagata accgttggcc tcaatcggcg
     6061 ttaaacccgc caccagatgg gcattaaacg agtatcccgg cagcagggga tcattttgcg
     6121 cttcagccat acttttcata ctcccgccat tcagagaaga aaccaattgt ccatattgca
     6181 tcagacattg ccgtcactgc gtcttttact ggctcttctc gctaaccaaa ccggtaaccc
     6241 cgcttattaa aagcattctg taacaaagcg ggaccaaagc catgacaaaa acgcgtaaca
     6301 aaagtgtcta taatcacggc agaaaagtcc acattgatta tttgcacggc gtcacacttt
     6361 gctatgccat agcattttta tccataagat tagcggatcc tacctgacgc tttttatcgc
     6421 aactctctac tgtttctcca tacccgtttt tttgggctag aaataatttt gtttaacttt
     6481 aagaaggaga tatacatatg gctagtcatc accatcacca tcaca
//
