LOCUS       sequence-132645-        7692 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express shRNA against mouse TNF-alpha.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7692)
  AUTHORS   Ogawa N, Kawai H, Terashima T, Kojima H, Oka K, Chan L, Maegawa H
  TITLE     Gene therapy for neuropathic pain by silencing of TNF-alpha 
            expression with lentiviral vectors targeting the dorsal root 
            ganglion in mice.
  JOURNAL   PLoS One. 2014 Mar 18;9(3):e92073. doi: 
            10.1371/journal.pone.0092073. eCollection 2014.
  PUBMED    24642694
REFERENCE   2  (bases 1 to 7692)
  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: "PLoS One.";
            date: "2014-03-18"; volume: "9(3):e92073. doi"; pages: " 
            10.1371/journal.pone.0092073. eCollection 2014"
COMMENT     Sequence Label: pLL3.7-TNFa-shRNA3
FEATURES             Location/Qualifiers
     source          1..7692
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(47..66)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        238..617
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        619..817
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     LTR             835..1015
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1062..1187
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1686..1919
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2104..2148
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    2446..2563
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2622..2935
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for mouse U6 snRNA (Das 
                     et al., 1988)"
     primer_bind     2854..2874
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     primer_bind     2994..3010
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(3052..3085)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     enhancer        3201..3504
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3505..3708
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     regulatory      3747..3756
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(3753..3755,3756..3758,3759..4472)
                     /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(3753..3755,3756..3758,3759..4469)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3798..3819)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4059..4078)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4406..4427
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(4492..4525)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    4581..5169
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4634..4654)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5052..5063)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5172..5188)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     LTR             5698..5878
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(5940..6528)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6020..6039)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6699..7490,7491..7559))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     7322..7341
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7560..7664)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg
       61 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt
      121 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc
      181 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac
      241 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat
      301 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
      361 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
      421 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
      481 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
      541 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
      601 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt
      661 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc
      721 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg
      781 gcggtaggcg tgtacggtgg gaggtctata taagcagcgc gttttgcctg tactgggtct
      841 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt
      901 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac
      961 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc
     1021 gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc
     1081 ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa
     1141 ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg
     1201 ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata
     1261 aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc
     1321 tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga
     1381 caggatcaga agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc
     1441 aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca
     1501 aaagtaagac caccgcacag caagcggccg gccgcgctga tcttcagacc tggaggagga
     1561 gatatgaggg acaattggag aagtgaatta tataaatata aagtagtaaa aattgaacca
     1621 ttaggagtag cacccaccaa ggcaaagaga agagtggtgc agagagaaaa aagagcagtg
     1681 ggaataggag ctttgttcct tgggttcttg ggagcagcag gaagcactat gggcgcagcg
     1741 tcaatgacgc tgacggtaca ggccagacaa ttattgtctg gtatagtgca gcagcagaac
     1801 aatttgctga gggctattga ggcgcaacag catctgttgc aactcacagt ctggggcatc
     1861 aagcagctcc aggcaagaat cctggctgtg gaaagatacc taaaggatca acagctcctg
     1921 gggatttggg gttgctctgg aaaactcatt tgcaccactg ctgtgccttg gaatgctagt
     1981 tggagtaata aatctctgga acagatttgg aatcacacga cctggatgga gtgggacaga
     2041 gaaattaaca attacacaag cttaatacac tccttaattg aagaatcgca aaaccagcaa
     2101 gaaaagaatg aacaagaatt attggaatta gataaatggg caagtttgtg gaattggttt
     2161 aacataacaa attggctgtg gtatataaaa ttattcataa tgatagtagg aggcttggta
     2221 ggtttaagaa tagtttttgc tgtactttct atagtgaata gagttaggca gggatattca
     2281 ccattatcgt ttcagaccca cctcccaacc ccgaggggac ccgacaggcc cgaaggaata
     2341 gaagaagaag gtggagagag agacagagac agatccattc gattagtgaa cggatcggca
     2401 ctgcgtgcgc caattctgca gacaaatggc agtattcatc cacaatttta aaagaaaagg
     2461 ggggattggg gggtacagtg caggggaaag aatagtagac ataatagcaa cagacataca
     2521 aactaaagaa ttacaaaaac aaattacaaa aattcaaaat tttcgggttt attacaggga
     2581 cagcagagat ccagtttggt tagtaccggg cccgctctag agatccgacg ccgccatctc
     2641 taggcccgcg ccggccccct cgcacagact tgtgggagaa gctcggctac tcccctgccc
     2701 cggttaattt gcatataata tttcctagta actatagagg cttaatgtgc gataaaagac
     2761 agataatctg ttctttttaa tactagctac attttacatg ataggcttgg atttctataa
     2821 gagatacaaa tactaaatta ttattttaaa aaacagcaca aaaggaaact caccctaact
     2881 gtaaagtaat tgtgtgtttt gagactataa atatcccttg gagaaaagcc ttgtttgcct
     2941 ccctctcatc agttctatct cgagatagaa ctgatgagag ggaggttttt tctcgaggtc
     3001 gacggtatcg ataagctcgc ttcacgagat tccagcaggt cgagggacct aataacttcg
     3061 tatagcatac attatacgaa gttatattaa gggttccaag cttaagcggc cgcgtggata
     3121 accgtattac cgccatgcat tagttattaa tagtaatcaa ttacggggtc attagttcat
     3181 agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg
     3241 cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata
     3301 gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta
     3361 catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc
     3421 gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca gtacatctac
     3481 gtattagtca tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga
     3541 tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg
     3601 ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg
     3661 caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagagctgg tttagtgaac
     3721 cgtcagatcc gctagcgcta ccggtcgcca ccatggtgag caagggcgag gagctgttca
     3781 ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg
     3841 tgtccggcga gggcgagggc gatgccacct acggcaagct gaccctgaag ttcatctgca
     3901 ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccctgacc tacggcgtgc
     3961 agtgcttcag ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc
     4021 ccgaaggcta cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc
     4081 gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg
     4141 acttcaagga ggacggcaac atcctggggc acaagctgga gtacaactac aacagccaca
     4201 acgtctatat catggccgac aagcagaaga acggcatcaa ggtgaacttc aagatccgcc
     4261 acaacatcga ggacggcagc gtgcagctcg ccgaccacta ccagcagaac acccccatcg
     4321 gcgacggccc cgtgctgctg cccgacaacc actacctgag cacccagtcc gccctgagca
     4381 aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga
     4441 tcactctcgg catggacgag ctgtacaagt aggaattcgt cgagggacct aataacttcg
     4501 tatagcatac attatacgaa gttatacatg tttaagggtt ccggttccac taggtacaat
     4561 tcgatatcaa gcttatcgat aatcaacctc tggattacaa aatttgtgaa agattgactg
     4621 gtattcttaa ctatgttgct ccttttacgc tatgtggata cgctgcttta atgcctttgt
     4681 atcatgctat tgcttcccgt atggctttca ttttctcctc cttgtataaa tcctggttgc
     4741 tgtctcttta tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt
     4801 ttgctgacgc aacccccact ggttggggca ttgccaccac ctgtcagctc ctttccggga
     4861 ctttcgcttt ccccctccct attgccacgg cggaactcat cgccgcctgc cttgcccgct
     4921 gctggacagg ggctcggctg ttgggcactg acaattccgt ggtgttgtcg gggaaatcat
     4981 cgtcctttcc ttggctgctc gcctgtgttg ccacctggat tctgcgcggg acgtccttct
     5041 gctacgtccc ttcggccctc aatccagcgg accttccttc ccgcggcctg ctgccggctc
     5101 tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc ctttgggccg
     5161 cctccccgca tcgataccgt cgacctcgat cgagacctag aaaaacatgg agcaatcaca
     5221 agtagcaata cagcagctac caatgctgat tgtgcctggc tagaagcaca agaggaggag
     5281 gaggtgggtt ttccagtcac acctcaggta cctttaagac caatgactta caaggcagct
     5341 gtagatctta gccacttttt aaaagaaaag gggggactgg aagggctaat tcactcccaa
     5401 cgaagacaag atatccttga tctgtggatc taccacacac aaggctactt ccctgattgg
     5461 cagaactaca caccagggcc agggatcaga tatccactga cctttggatg gtgctacaag
     5521 ctagtaccag ttgagcaaga gaaggtagaa gaagccaatg aaggagagaa cacccgcttg
     5581 ttacaccctg tgagcctgca tgggatggat gacccggaga gagaagtatt agagtggagg
     5641 tttgacagcc gcctagcatt tcatcacatg gcccgagagc tgcatccgga ctgtactggg
     5701 tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg
     5761 cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt
     5821 gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagc
     5881 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     5941 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     6001 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     6061 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     6121 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     6181 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     6241 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     6301 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     6361 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
     6421 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     6481 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     6541 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     6601 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     6661 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     6721 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     6781 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     6841 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     6901 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     6961 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     7021 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     7081 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     7141 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     7201 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     7261 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     7321 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     7381 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     7441 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     7501 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     7561 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     7621 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     7681 gtgccacctg ac
//
