LOCUS       sequence-343870-        9613 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  All-in-one huMDA5 rescue-MDA5 shRNA knockdown vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9613)
  AUTHORS   Guney MH, Nagalekshmi K, McCauley SM, Carbone C, Aydemir O, Luban J
  TITLE     IFIH1 (MDA5) is required for innate immune detection of 
            intron-containing RNA expressed from the HIV-1 provirus.
  JOURNAL   Proc Natl Acad Sci U S A. 2024 Jul 16;121(29):e2404349121. doi: 
            10.1073/pnas.2404349121. Epub 2024 Jul 10.
  PUBMED    38985764
REFERENCE   2  (bases 1 to 9613)
  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: "Proc Natl 
            Acad Sci U S A."; date: "2024-07-16"; volume: "121(29):e2404349121. 
            doi"; pages: " 10.1073/pnas.2404349121. Epub 2024 Jul 10"
COMMENT     Sequence Label: pPU-huMDA5-miR30-MDA5
FEATURES             Location/Qualifiers
     source          1..9613
                     /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"
     primer_bind     complement(240..260)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        245..574
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      425..560
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     487..506
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     enhancer        586..965
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        967..1165
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1120..1140
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             1183..1363
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1410..1535
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    2032..2265
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2450..2494
                     /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    2792..2909
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        3046..3453
                     /label=SFFV promoter
                     /note="spleen focus-forming virus long terminal repeat 
                     (LTR) promoter"
     CDS             3468..4064
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(3468..3487)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3964..3984
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             4065..4292
                     /codon_start=1
                     /product="human ubiquitin variant in which Lys-48 has been 
                     mutated to arginine"
                     /label=ubiquitin-K48R
                     /translation="MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIF
                     AGRQLEDGRTLSDYNIQKESTLHLVLRLRGG"
     ncRNA           7462..7476
                     /label=miR-30a loop
                     /note="loop from the the 71-nt precursor of the human 
                     miR-30a microRNA (Zeng et al., 2002)"
                     /ncRNA_class="miRNA"
     LTR             7610..7790
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(7861..8449)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7941..7960)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(8620..9411,9412..9480))
                     /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     9243..9262
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(9481..9585)
                     /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 acgcgctgtg
      241 gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca
      301 aagcatgcat ctcaattagt cagcaaccag gtgtggaaag tccccaggct ccccagcagg
      361 cagaagtatg caaagcatgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc
      421 gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat
      481 tttttttatt tatgcagagg ccgaggccgc ctctgcctct gagctattcc agaagtagtg
      541 aggaggcttt tttggaggcc taggcttttg caaaaagctc gcgttgacat tgattattga
      601 ctagttatta atagtaatca attacggggt cattagttca tagcccatat atggagttcc
      661 gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat
      721 tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc
      781 aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc
      841 caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt
      901 acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta
      961 ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg
     1021 gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac
     1081 gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg
     1141 tacggtggga ggtctatata agcagcgcgt tttgcctgta ctgggtctct ctggttagac
     1201 cagatctgag cctgggagct ctctggctaa ctagggaacc cactgcttaa gcctcaataa
     1261 agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag
     1321 agatccctca gaccctttta gtcagtgtgg aaaatctcta gcagtggcgc ccgaacaggg
     1381 acttgaaagc gaaagggaaa ccagaggagc tctctcgacg caggactcgg cttgctgaag
     1441 cgcgcacggc aagaggcgag gggcggcgac tggtgagtac gccaaaaatt ttgactagcg
     1501 gaggctagaa ggagagagat gggtgcgaga gcgtcagtat taagcggggg agaattagat
     1561 cgcgatggga aaaaattcgg ttaaggccag ggggaaagaa aaaatataaa ttaaaacata
     1621 tagtatgggc aagcagggag ctagaacgat tcgcagttaa tcctggcctg ttagaaacat
     1681 cagaaggctg tagacaaata ctgggacagc tacaaccatc ccttcagaca ggatcagaag
     1741 aacttagatc attatataat acagtagcaa ccctctattg tgtgcatcaa aggatagaga
     1801 taaaagacac caaggaagct ttagacaaga tagaggaaga gcaaaacaaa agtaagacca
     1861 ccgcacagca agcggccggc cgctgatctt cagacctgga ggaggagata tgagggacaa
     1921 ttggagaagt gaattatata aatataaagt agtaaaaatt gaaccattag gagtagcacc
     1981 caccaaggca aagagaagag tggtgcagag agaaaaaaga gcagtgggaa taggagcttt
     2041 gttccttggg ttcttgggag cagcaggaag cactatgggc gcagcgtcaa tgacgctgac
     2101 ggtacaggcc agacaattat tgtctggtat agtgcagcag cagaacaatt tgctgagggc
     2161 tattgaggcg caacagcatc tgttgcaact cacagtctgg ggcatcaagc agctccaggc
     2221 aagaatcctg gctgtggaaa gatacctaaa ggatcaacag ctcctgggga tttggggttg
     2281 ctctggaaaa ctcatttgca ccactgctgt gccttggaat gctagttgga gtaataaatc
     2341 tctggaacag atttggaatc acacgacctg gatggagtgg gacagagaaa ttaacaatta
     2401 cacaagctta atacactcct taattgaaga atcgcaaaac cagcaagaaa agaatgaaca
     2461 agaattattg gaattagata aatgggcaag tttgtggaat tggtttaaca taacaaattg
     2521 gctgtggtat ataaaattat tcataatgat agtaggaggc ttggtaggtt taagaatagt
     2581 ttttgctgta ctttctatag tgaatagagt taggcaggga tattcaccat tatcgtttca
     2641 gacccacctc ccaaccccga ggggacccga caggcccgaa ggaatagaag aagaaggtgg
     2701 agagagagac agagacagat ccattcgatt agtgaacgga tcggcactgc gtgcgccaat
     2761 tctgcagaca aatggcagta ttcatccaca attttaaaag aaaagggggg attggggggt
     2821 acagtgcagg ggaaagaata gtagacataa tagcaacaga catacaaact aaagaattac
     2881 aaaaacaaat tacaaaaatt caaaattttc gggtttatta cagggacagc agagatccag
     2941 tttggttaat taactgcagc cccgataaaa taaaagattt tatttagtct ccagaaaaag
     3001 gggggaatga aagaccccac ctgtaggttt ggcaagctag ctgcagtaac gccattttgc
     3061 aaggcatgga aaaataccaa accaagaata gagaagttca gatcaagggc gggtacatga
     3121 aaatagctaa cgttgggcca aacaggatat ctgcggtgag cagtttcggc cccggcccgg
     3181 ggccaagaac agatggtcac cgcagtttcg gccccggccc gaggccaaga acagatggtc
     3241 cccagatatg gcccaaccct cagcagtttc ttaagaccca tcagatgttt ccaggctccc
     3301 ccaaggacct gaaatgaccc tgcgccttat ttgaattaac caatcagcct gcttctcgct
     3361 tctgttcgcg cgcttctgct tcccgagctc tataaaagag ctcacaaccc ctcactcggc
     3421 gcgccagtcc tccgacagac tgagtcgccc gggggtctag tgccaccatg accgagtaca
     3481 agcccacggt gcgcctcgcc acccgcgacg acgtcccccg ggccgtacgc accctcgccg
     3541 ccgcgttcgc cgactacccc gccacgcgcc acaccgtgga cccggaccgc cacatcgagc
     3601 gggtcaccga gctgcaagaa ctcttcctca cgcgcgtcgg gctcgacatc ggcaaggtgt
     3661 gggtcgcgga cgacggcgcc gcggtggcgg tctggaccac gccggagagc gtcgaagcgg
     3721 gggcggtgtt cgccgagatc ggcccgcgca tggccgagtt gagcggttcc cggctggccg
     3781 cgcagcaaca gatggaaggc ctcctggcgc cgcaccggcc caaggagccc gcgtggttcc
     3841 tggccaccgt cggcgtctcg cccgaccacc agggcaaggg tctgggcagc gccgtcgtgc
     3901 tccccggagt ggaggcggcc gagcgcgccg gggtgcccgc cttcctggag acctccgcgc
     3961 cccgcaacct ccccttctac gagcggctcg gcttcaccgt caccgccgac gtcgaggtgc
     4021 ccgaaggacc gcgcacctgg tgcatgaccc gcaagcccgg tgccatgcag atctttgtga
     4081 agaccctcac tggcaaaacc atcacccttg aggtcgagcc cagtgacacc attgagaatg
     4141 tcaaagccaa aattcaagac aaggagggta tcccacctga ccagcagcgt ctgatatttg
     4201 ccggcagaca gctggaggat ggccgcactc tctcagacta caacatccag aaagagtcca
     4261 ccctgcacct ggtgttgcgc ctgcgaggtg gcggatccat gagtaatggt tactcaaccg
     4321 atgagaattt caggtatctg atttcctgtt tcagagcccg agtcaaaatg tacatacaag
     4381 tcgaacctgt gttggattat ctcacttttc tcccagcaga agtgaaggaa caaattcaac
     4441 ggacggtagc aacttcagga aatatgcagg cggttgagct tttactcagc acattggaaa
     4501 agggtgtgtg gcaccttggg tggactagag agtttgtaga ggccctccgc cgcactggta
     4561 gccccttggc tgcgcgatac atgaacccgg agctgaccga tttgccatca ccttcctttg
     4621 agaacgccca tgatgagtat ctgcaactct tgaaccttct tcaaccgacc ttggttgaca
     4681 aactgttggt acgcgacgta ctggacaaat gtatggagga agaactgctg accattgaag
     4741 acaggaatcg gatcgccgcc gccgagaata acggcaatga atccggcgtc agggaattgc
     4801 tgaaacgcat cgttcaaaag gagaactggt ttagtgcctt tctcaacgtg ctgcgccaaa
     4861 ccggtaataa tgagttggta caagagttga cgggttccga ctgttccgag tcaaatgcgg
     4921 aaatagaaaa cctttctcaa gtagacggcc cgcaggtcga ggaacagctt ctgagtacga
     4981 cagtccaacc aaatctggag aaggaggtgt ggggaatgga aaataacagt tctgaatcaa
     5041 gtttcgccga cagttcagtc gtgtccgaat ctgacacgag cttggcagaa ggatcagtta
     5101 gttgtttgga cgagtctctt gggcacaact ccaacatggg cagcgactct gggactatgg
     5161 gtagcgactc tgatgaggaa aacgtggccg ctcgggcgtc cccagagcct gaacttcagc
     5221 ttcgccccta tcagatggag gttgcgcagc ccgccttgga aggtaaaaac atcattatct
     5281 gtttgcctac cggcagtgga aagacacgag tagcggtcta catcgccaaa gaccacttgg
     5341 acaaaaagaa aaaggcgtca gagcctggta aagtgatagt tttggtcaac aaggttctct
     5401 tggtggagca gctctttcgg aaggagtttc agccctttct gaagaaatgg tatcgagtga
     5461 tcggcttgtc aggcgatacg cagttgaaaa tatcatttcc agaggtagta aagtcctgtg
     5521 acatcatcat atcaactgcc caaattcttg aaaattcctt gctgaacttg gaaaacgggg
     5581 aagacgcggg ggtacaactc agcgatttca gtctgatcat cattgatgag tgtcatcaca
     5641 cgaataaaga agcagtgtat aacaatataa tgaggcacta tctgatgcag aaacttaaaa
     5701 acaacagact gaaaaaggag aacaagcccg tcattccatt gccgcaaatt cttggcctta
     5761 cggccagccc cggcgttggt ggggccacca aacaagccaa ggctgaagaa cacatcctca
     5821 aattgtgcgc aaacttggat gcttttacaa taaagacagt gaaagaaaat cttgaccagc
     5881 tcaaaaacca gatacaagaa ccttgcaaga aattcgcgat agccgacgct acccgagaag
     5941 atccttttaa agagaagctg ctggaaatta tgacgcggat acagacgtat tgtcaaatga
     6001 gccctatgtc tgactttggg acgcaaccat atgagcaatg ggccatacag atggaaaaaa
     6061 aagctgcgaa agagggcaat cgaaaagagc gcgtctgtgc ggagcatctc aggaagtata
     6121 atgaggcgct gcagatcaac gatacgatcc gcatgatcga tgcgtacact cacctggaaa
     6181 cgttttacaa tgaagagaaa gataagaaat ttgcggtcat cgaggacgac tcagatgaag
     6241 gcggtgacga cgagtattgc gacggagatg aggacgagga tgatcttaag aagcctttga
     6301 aactcgatga gactgaccgc ttcctcatga cccttttctt cgagaataat aaaatgctga
     6361 agaggctggc cgaaaatcca gagtacgaga acgagaagct cacgaaactg cgcaatacaa
     6421 taatggaaca atatacccgc acggaagaaa gcgcaagagg catcattttc actaaaacca
     6481 ggcagagtgc ctacgccttg agtcagtgga taaccgaaaa tgagaaattc gctgaggtcg
     6541 gtgtaaaagc tcaccatctt atcggcgctg gacacagttc agagtttaaa ccgatgactc
     6601 aaaatgagca gaaagaggta attagtaaat tccgaacagg aaagattaac ttgctgatag
     6661 ccacaactgt ggccgaagag ggcttggaca taaaggagtg caatatcgtc atacggtatg
     6721 gtctcgtcac taacgaaata gcaatggtcc aggcccgcgg acgggctaga gccgatgaga
     6781 gcacttatgt acttgttgcc cattctggaa gcggggtgat cgagcatgaa accgtgaatg
     6841 attttcgcga aaaaatgatg tacaaggcta tacactgtgt gcaaaacatg aagccagaag
     6901 aatacgccca caagattctg gaactccaaa tgcaaagcat tatggagaaa aaaatgaaga
     6961 ctaaacgcaa cattgcgaaa cattacaaga acaatccatc tctgattaca tttctttgta
     7021 agaactgctc cgtgctggcc tgttctggag aggacattca cgtaatagag aaaatgcacc
     7081 acgtgaatat gactcctgag tttaaggagc tttacatagt tcgagagaac aaggcgctcc
     7141 aaaagaagtg tgcagattat caaataaacg gagaaatcat ttgtaagtgt ggtcaagcgt
     7201 ggggaaccat gatggtgcac aaaggcctcg atcttccctg cttgaaaata agaaattttg
     7261 tagttgtatt caaaaacaac tccactaaaa aacaatataa gaaatgggta gaactgccaa
     7321 ttacgttccc taacttggac tatagtgaat gttgtctgtt tagtgacgaa gattaggcgg
     7381 ccgccttctt aacccaacag aaggctcgag aaggtatatt gctgttgaca gtgagcgacg
     7441 agagaagatg atgtataaat agtgaagcca cagatgtatt tatacatcat cttctctcgg
     7501 tgcctactgc ctcggacttc aaggggctag aattcggcag ctgtagatct tagccacttt
     7561 ttaaaagaaa aggggggact ggaagggcta actgcatccg gactgtactg ggtctctctg
     7621 gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc
     7681 tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg
     7741 taactagaga tccctcagac ccttttagtc agtgtggaaa atctctagca gggcccgttt
     7801 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     7861 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     7921 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     7981 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     8041 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     8101 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     8161 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     8221 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     8281 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
     8341 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     8401 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     8461 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     8521 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
     8581 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
     8641 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
     8701 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
     8761 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
     8821 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     8881 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
     8941 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     9001 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     9061 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     9121 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     9181 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
     9241 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     9301 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     9361 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     9421 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
     9481 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
     9541 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     9601 agtgccacct gac
//
