LOCUS       sequence-343778-        9613 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  All-in-one huMDA5 rescue-control 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-L1221
FEATURES             Location/Qualifiers
     source          1..9613
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        62..441
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        443..641
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     596..616
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             659..839
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    886..1011
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1508..1741
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1926..1970
                     /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    2268..2385
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2522..2929
                     /label=SFFV promoter
                     /note="spleen focus-forming virus long terminal repeat 
                     (LTR) promoter"
     CDS             2944..3540
                     /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(2944..2963)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3440..3460
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             3541..3768
                     /codon_start=1
                     /product="human ubiquitin variant in which Lys-48 has been 
                     mutated to arginine"
                     /label=ubiquitin-K48R
                     /translation="MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIF
                     AGRQLEDGRTLSDYNIQKESTLHLVLRLRGG"
     ncRNA           6938..6952
                     /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             7086..7266
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(7337..7925)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7417..7436)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(8096..8887,8888..8956))
                     /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     8719..8738
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8957..9061)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(9136..9155)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(9329..9349)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        join(9334..9613,1..50)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      join(9514..9613,1..36)
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     9576..9595
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
ORIGIN
        1 tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa aagctcgcgt
       61 tgacattgat tattgactag ttattaatag taatcaatta cggggtcatt agttcatagc
      121 ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc
      181 aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg
      241 actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat
      301 caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc
      361 tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta catctacgta
      421 ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag
      481 cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt
      541 tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa
      601 atgggcggta ggcgtgtacg gtgggaggtc tatataagca gcgcgttttg cctgtactgg
      661 gtctctctgg ttagaccaga tctgagcctg ggagctctct ggctaactag ggaacccact
      721 gcttaagcct caataaagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg
      781 tgactctggt aactagagat ccctcagacc cttttagtca gtgtggaaaa tctctagcag
      841 tggcgcccga acagggactt gaaagcgaaa gggaaaccag aggagctctc tcgacgcagg
      901 actcggcttg ctgaagcgcg cacggcaaga ggcgaggggc ggcgactggt gagtacgcca
      961 aaaattttga ctagcggagg ctagaaggag agagatgggt gcgagagcgt cagtattaag
     1021 cgggggagaa ttagatcgcg atgggaaaaa attcggttaa ggccaggggg aaagaaaaaa
     1081 tataaattaa aacatatagt atgggcaagc agggagctag aacgattcgc agttaatcct
     1141 ggcctgttag aaacatcaga aggctgtaga caaatactgg gacagctaca accatccctt
     1201 cagacaggat cagaagaact tagatcatta tataatacag tagcaaccct ctattgtgtg
     1261 catcaaagga tagagataaa agacaccaag gaagctttag acaagataga ggaagagcaa
     1321 aacaaaagta agaccaccgc acagcaagcg gccggccgct gatcttcaga cctggaggag
     1381 gagatatgag ggacaattgg agaagtgaat tatataaata taaagtagta aaaattgaac
     1441 cattaggagt agcacccacc aaggcaaaga gaagagtggt gcagagagaa aaaagagcag
     1501 tgggaatagg agctttgttc cttgggttct tgggagcagc aggaagcact atgggcgcag
     1561 cgtcaatgac gctgacggta caggccagac aattattgtc tggtatagtg cagcagcaga
     1621 acaatttgct gagggctatt gaggcgcaac agcatctgtt gcaactcaca gtctggggca
     1681 tcaagcagct ccaggcaaga atcctggctg tggaaagata cctaaaggat caacagctcc
     1741 tggggatttg gggttgctct ggaaaactca tttgcaccac tgctgtgcct tggaatgcta
     1801 gttggagtaa taaatctctg gaacagattt ggaatcacac gacctggatg gagtgggaca
     1861 gagaaattaa caattacaca agcttaatac actccttaat tgaagaatcg caaaaccagc
     1921 aagaaaagaa tgaacaagaa ttattggaat tagataaatg ggcaagtttg tggaattggt
     1981 ttaacataac aaattggctg tggtatataa aattattcat aatgatagta ggaggcttgg
     2041 taggtttaag aatagttttt gctgtacttt ctatagtgaa tagagttagg cagggatatt
     2101 caccattatc gtttcagacc cacctcccaa ccccgagggg acccgacagg cccgaaggaa
     2161 tagaagaaga aggtggagag agagacagag acagatccat tcgattagtg aacggatcgg
     2221 cactgcgtgc gccaattctg cagacaaatg gcagtattca tccacaattt taaaagaaaa
     2281 ggggggattg gggggtacag tgcaggggaa agaatagtag acataatagc aacagacata
     2341 caaactaaag aattacaaaa acaaattaca aaaattcaaa attttcgggt ttattacagg
     2401 gacagcagag atccagtttg gttaattaac tgcagccccg ataaaataaa agattttatt
     2461 tagtctccag aaaaaggggg gaatgaaaga ccccacctgt aggtttggca agctagctgc
     2521 agtaacgcca ttttgcaagg catggaaaaa taccaaacca agaatagaga agttcagatc
     2581 aagggcgggt acatgaaaat agctaacgtt gggccaaaca ggatatctgc ggtgagcagt
     2641 ttcggccccg gcccggggcc aagaacagat ggtcaccgca gtttcggccc cggcccgagg
     2701 ccaagaacag atggtcccca gatatggccc aaccctcagc agtttcttaa gacccatcag
     2761 atgtttccag gctcccccaa ggacctgaaa tgaccctgcg ccttatttga attaaccaat
     2821 cagcctgctt ctcgcttctg ttcgcgcgct tctgcttccc gagctctata aaagagctca
     2881 caacccctca ctcggcgcgc cagtcctccg acagactgag tcgcccgggg gtctagtgcc
     2941 accatgaccg agtacaagcc cacggtgcgc ctcgccaccc gcgacgacgt cccccgggcc
     3001 gtacgcaccc tcgccgccgc gttcgccgac taccccgcca cgcgccacac cgtggacccg
     3061 gaccgccaca tcgagcgggt caccgagctg caagaactct tcctcacgcg cgtcgggctc
     3121 gacatcggca aggtgtgggt cgcggacgac ggcgccgcgg tggcggtctg gaccacgccg
     3181 gagagcgtcg aagcgggggc ggtgttcgcc gagatcggcc cgcgcatggc cgagttgagc
     3241 ggttcccggc tggccgcgca gcaacagatg gaaggcctcc tggcgccgca ccggcccaag
     3301 gagcccgcgt ggttcctggc caccgtcggc gtctcgcccg accaccaggg caagggtctg
     3361 ggcagcgccg tcgtgctccc cggagtggag gcggccgagc gcgccggggt gcccgccttc
     3421 ctggagacct ccgcgccccg caacctcccc ttctacgagc ggctcggctt caccgtcacc
     3481 gccgacgtcg aggtgcccga aggaccgcgc acctggtgca tgacccgcaa gcccggtgcc
     3541 atgcagatct ttgtgaagac cctcactggc aaaaccatca cccttgaggt cgagcccagt
     3601 gacaccattg agaatgtcaa agccaaaatt caagacaagg agggtatccc acctgaccag
     3661 cagcgtctga tatttgccgg cagacagctg gaggatggcc gcactctctc agactacaac
     3721 atccagaaag agtccaccct gcacctggtg ttgcgcctgc gaggtggcgg atccatgagt
     3781 aatggttact caaccgatga gaatttcagg tatctgattt cctgtttcag agcccgagtc
     3841 aaaatgtaca tacaagtcga acctgtgttg gattatctca cttttctccc agcagaagtg
     3901 aaggaacaaa ttcaacggac ggtagcaact tcaggaaata tgcaggcggt tgagctttta
     3961 ctcagcacat tggaaaaggg tgtgtggcac cttgggtgga ctagagagtt tgtagaggcc
     4021 ctccgccgca ctggtagccc cttggctgcg cgatacatga acccggagct gaccgatttg
     4081 ccatcacctt cctttgagaa cgcccatgat gagtatctgc aactcttgaa ccttcttcaa
     4141 ccgaccttgg ttgacaaact gttggtacgc gacgtactgg acaaatgtat ggaggaagaa
     4201 ctgctgacca ttgaagacag gaatcggatc gccgccgccg agaataacgg caatgaatcc
     4261 ggcgtcaggg aattgctgaa acgcatcgtt caaaaggaga actggtttag tgcctttctc
     4321 aacgtgctgc gccaaaccgg taataatgag ttggtacaag agttgacggg ttccgactgt
     4381 tccgagtcaa atgcggaaat agaaaacctt tctcaagtag acggcccgca ggtcgaggaa
     4441 cagcttctga gtacgacagt ccaaccaaat ctggagaagg aggtgtgggg aatggaaaat
     4501 aacagttctg aatcaagttt cgccgacagt tcagtcgtgt ccgaatctga cacgagcttg
     4561 gcagaaggat cagttagttg tttggacgag tctcttgggc acaactccaa catgggcagc
     4621 gactctggga ctatgggtag cgactctgat gaggaaaacg tggccgctcg ggcgtcccca
     4681 gagcctgaac ttcagcttcg cccctatcag atggaggttg cgcagcccgc cttggaaggt
     4741 aaaaacatca ttatctgttt gcctaccggc agtggaaaga cacgagtagc ggtctacatc
     4801 gccaaagacc acttggacaa aaagaaaaag gcgtcagagc ctggtaaagt gatagttttg
     4861 gtcaacaagg ttctcttggt ggagcagctc tttcggaagg agtttcagcc ctttctgaag
     4921 aaatggtatc gagtgatcgg cttgtcaggc gatacgcagt tgaaaatatc atttccagag
     4981 gtagtaaagt cctgtgacat catcatatca actgcccaaa ttcttgaaaa ttccttgctg
     5041 aacttggaaa acggggaaga cgcgggggta caactcagcg atttcagtct gatcatcatt
     5101 gatgagtgtc atcacacgaa taaagaagca gtgtataaca atataatgag gcactatctg
     5161 atgcagaaac ttaaaaacaa cagactgaaa aaggagaaca agcccgtcat tccattgccg
     5221 caaattcttg gccttacggc cagccccggc gttggtgggg ccaccaaaca agccaaggct
     5281 gaagaacaca tcctcaaatt gtgcgcaaac ttggatgctt ttacaataaa gacagtgaaa
     5341 gaaaatcttg accagctcaa aaaccagata caagaacctt gcaagaaatt cgcgatagcc
     5401 gacgctaccc gagaagatcc ttttaaagag aagctgctgg aaattatgac gcggatacag
     5461 acgtattgtc aaatgagccc tatgtctgac tttgggacgc aaccatatga gcaatgggcc
     5521 atacagatgg aaaaaaaagc tgcgaaagag ggcaatcgaa aagagcgcgt ctgtgcggag
     5581 catctcagga agtataatga ggcgctgcag atcaacgata cgatccgcat gatcgatgcg
     5641 tacactcacc tggaaacgtt ttacaatgaa gagaaagata agaaatttgc ggtcatcgag
     5701 gacgactcag atgaaggcgg tgacgacgag tattgcgacg gagatgagga cgaggatgat
     5761 cttaagaagc ctttgaaact cgatgagact gaccgcttcc tcatgaccct tttcttcgag
     5821 aataataaaa tgctgaagag gctggccgaa aatccagagt acgagaacga gaagctcacg
     5881 aaactgcgca atacaataat ggaacaatat acccgcacgg aagaaagcgc aagaggcatc
     5941 attttcacta aaaccaggca gagtgcctac gccttgagtc agtggataac cgaaaatgag
     6001 aaattcgctg aggtcggtgt aaaagctcac catcttatcg gcgctggaca cagttcagag
     6061 tttaaaccga tgactcaaaa tgagcagaaa gaggtaatta gtaaattccg aacaggaaag
     6121 attaacttgc tgatagccac aactgtggcc gaagagggct tggacataaa ggagtgcaat
     6181 atcgtcatac ggtatggtct cgtcactaac gaaatagcaa tggtccaggc ccgcggacgg
     6241 gctagagccg atgagagcac ttatgtgctt gttgcccatt ctggaagcgg ggtgatcgag
     6301 catgaaaccg tgaatgattt tcgcgaaaaa atgatgtaca aggctataca ctgtgtgcaa
     6361 aacatgaagc cagaagaata cgcccacaag attctggaac tccaaatgca aagcattatg
     6421 gagaaaaaaa tgaagactaa acgcaacatt gcgaaacatt acaagaacaa tccatctctg
     6481 attacatttc tttgtaagaa ctgctccgtg ctggcctgtt ctggagagga cattcacgta
     6541 atagagaaaa tgcaccacgt gaatatgact cctgagttta aggagcttta catagttcga
     6601 gagaacaagg cgctccaaaa gaagtgtgca gattatcaaa taaacggaga aatcatttgt
     6661 aagtgtggtc aagcgtgggg aaccatgatg gtgcacaaag gcctcgatct tccctgcttg
     6721 aaaataagaa attttgtagt tgtattcaaa aacaactcca ctaaaaaaca atataagaaa
     6781 tgggtagaac tgccaattac gttccctaac ttggactata gtgaatgttg tctgtttagt
     6841 gacgaagatt aggcggccgc cttcttaacc caacagaagg ctcgagaagg tatattgctg
     6901 ttgacagtga gcgcacaaac gctctcatcg acaagtagtg aagccacaga tgtacttgtc
     6961 gatgagagcg tttgtatgcc tactgcctcg gacttcaagg ggctagaatt cggcagctgt
     7021 agatcttagc cactttttaa aagaaaaggg gggactggaa gggctaactg catccggact
     7081 gtactgggtc tctctggtta gaccagatct gagcctggga gctctctggc taactaggga
     7141 acccactgct taagcctcaa taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc
     7201 tgttgtgtga ctctggtaac tagagatccc tcagaccctt ttagtcagtg tggaaaatct
     7261 ctagcagggc ccgtttcatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     7321 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     7381 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     7441 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     7501 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     7561 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     7621 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     7681 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     7741 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     7801 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     7861 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     7921 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     7981 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     8041 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     8101 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     8161 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     8221 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     8281 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     8341 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     8401 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     8461 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     8521 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     8581 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     8641 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     8701 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     8761 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     8821 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     8881 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     8941 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     9001 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     9061 gcacatttcc ccgaaaagtg ccacctgacg tcgacggatc gggagatctc ccgatcccct
     9121 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt atctgctccc
     9181 tgcttgtgtg ttggaggtcg ctgagtagtg cgcgagcaaa atttaagcta caacaaggca
     9241 aggcttgacc gacaattgca tgaagaatct gcttagggtt aggcgttttg cgctgcttcg
     9301 cgatgtacgg gccagatata cgcgctgtgg aatgtgtgtc agttagggtg tggaaagtcc
     9361 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccagg
     9421 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag
     9481 tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc
     9541 gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc
     9601 tctgcctctg agc
//
