LOCUS       sequence-343750-        6772 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  All-in-one control rescue-MDA5 shRNA knockdown vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6772)
  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 6772)
  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-HSA-miR30-MDA5
FEATURES             Location/Qualifiers
     source          1..6772
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     400..420
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     ncRNA           1057..1071
                     /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             1205..1385
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(1456..2044)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1536..1555)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2215..3006,3007..3075))
                     /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     2838..2857
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3076..3180)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(3255..3274)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3448..3468)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3453..3782
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3633..3768
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3695..3714
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     enhancer        3794..4173
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4175..4373
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4328..4348
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             4391..4571
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    4618..4743
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    5240..5473
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             5658..5702
                     /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    6000..6117
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        6254..6661
                     /label=SFFV promoter
                     /note="spleen focus-forming virus long terminal repeat 
                     (LTR) promoter"
     CDS             join(6676..6772,1..500)
                     /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(6676..6695)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
ORIGIN
        1 cgcgccacac cgtggacccg gaccgccaca tcgagcgggt caccgagctg caagaactct
       61 tcctcacgcg cgtcgggctc gacatcggca aggtgtgggt cgcggacgac ggcgccgcgg
      121 tggcggtctg gaccacgccg gagagcgtcg aagcgggggc ggtgttcgcc gagatcggcc
      181 cgcgcatggc cgagttgagc ggttcccggc tggccgcgca gcaacagatg gaaggcctcc
      241 tggcgccgca ccggcccaag gagcccgcgt ggttcctggc caccgtcggc gtctcgcccg
      301 accaccaggg caagggtctg ggcagcgccg tcgtgctccc cggagtggag gcggccgagc
      361 gcgccggggt gcccgccttc ctggagacct ccgcgccccg caacctcccc ttctacgagc
      421 ggctcggctt caccgtcacc gccgacgtcg aggtgcccga aggaccgcgc acctggtgca
      481 tgacccgcaa gcccggtgcc atgcagatct ttgtgaagac cctcactggc aaaaccatca
      541 cccttgaggt cgagcccagt gacaccattg agaatgtcaa agccaaaatt caagacaagg
      601 agggtatccc acctgaccag cagcgtctga tatttgccgg cagacagctg gaggatggcc
      661 gcactctctc agactacaac atccagaaag agtccaccct gcacctggtg ctgagcctgc
      721 gaggtggcat gggcagagct atggtggcca ggctagggct ggggttgctg cttctggcac
      781 tgctcctacc cacacagatt tactgcaacc aaacatctgt tgcacccttt cctggcaacc
      841 agaatatctc tgcttcccca aatccaagta atgctaccac cagagggggt ggcagctccc
      901 tccagtccac agctggtctg ctggcactgt ccctcagcct gctacatctg tactgttgat
      961 ctagaggatc cgcggccgcc ttcttaaccc aacagaaggc tcgagaaggt atattgctgt
     1021 tgacagtgag cgacgagaga agatgatgta taaatagtga agccacagat gtatttatac
     1081 atcatcttct ctcggtgcct actgcctcgg acttcaaggg gctagaattc ggcagctgta
     1141 gatcttagcc actttttaaa agaaaagggg ggactggaag ggctaactgc atccggactg
     1201 tactgggtct ctctggttag accagatctg agcctgggag ctctctggct aactagggaa
     1261 cccactgctt aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct
     1321 gttgtgtgac tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc
     1381 tagcagggcc cgtttcatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     1441 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     1501 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     1561 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     1621 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     1681 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     1741 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     1801 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     1861 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
     1921 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     1981 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     2041 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     2101 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     2161 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     2221 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     2281 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     2341 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     2401 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     2461 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     2521 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     2581 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     2641 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     2701 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     2761 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     2821 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     2881 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     2941 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     3001 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     3061 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
     3121 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     3181 cacatttccc cgaaaagtgc cacctgacgt cgacggatcg ggagatctcc cgatccccta
     3241 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta tctgctccct
     3301 gcttgtgtgt tggaggtcgc tgagtagtgc gcgagcaaaa tttaagctac aacaaggcaa
     3361 ggcttgaccg acaattgcat gaagaatctg cttagggtta ggcgttttgc gctgcttcgc
     3421 gatgtacggg ccagatatac gcgctgtgga atgtgtgtca gttagggtgt ggaaagtccc
     3481 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt
     3541 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt
     3601 cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg
     3661 cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct
     3721 ctgcctctga gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca
     3781 aaaagctcgc gttgacattg attattgact agttattaat agtaatcaat tacggggtca
     3841 ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct
     3901 ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta
     3961 acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac
     4021 ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt
     4081 aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag
     4141 tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat
     4201 gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat
     4261 gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc
     4321 ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag cagcgcgttt
     4381 tgcctgtact gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact
     4441 agggaaccca ctgcttaagc ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc
     4501 ccgtctgttg tgtgactctg gtaactagag atccctcaga cccttttagt cagtgtggaa
     4561 aatctctagc agtggcgccc gaacagggac ttgaaagcga aagggaaacc agaggagctc
     4621 tctcgacgca ggactcggct tgctgaagcg cgcacggcaa gaggcgaggg gcggcgactg
     4681 gtgagtacgc caaaaatttt gactagcgga ggctagaagg agagagatgg gtgcgagagc
     4741 gtcagtatta agcgggggag aattagatcg cgatgggaaa aaattcggtt aaggccaggg
     4801 ggaaagaaaa aatataaatt aaaacatata gtatgggcaa gcagggagct agaacgattc
     4861 gcagttaatc ctggcctgtt agaaacatca gaaggctgta gacaaatact gggacagcta
     4921 caaccatccc ttcagacagg atcagaagaa cttagatcat tatataatac agtagcaacc
     4981 ctctattgtg tgcatcaaag gatagagata aaagacacca aggaagcttt agacaagata
     5041 gaggaagagc aaaacaaaag taagaccacc gcacagcaag cggccggccg ctgatcttca
     5101 gacctggagg aggagatatg agggacaatt ggagaagtga attatataaa tataaagtag
     5161 taaaaattga accattagga gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag
     5221 aaaaaagagc agtgggaata ggagctttgt tccttgggtt cttgggagca gcaggaagca
     5281 ctatgggcgc agcgtcaatg acgctgacgg tacaggccag acaattattg tctggtatag
     5341 tgcagcagca gaacaatttg ctgagggcta ttgaggcgca acagcatctg ttgcaactca
     5401 cagtctgggg catcaagcag ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg
     5461 atcaacagct cctggggatt tggggttgct ctggaaaact catttgcacc actgctgtgc
     5521 cttggaatgc tagttggagt aataaatctc tggaacagat ttggaatcac acgacctgga
     5581 tggagtggga cagagaaatt aacaattaca caagcttaat acactcctta attgaagaat
     5641 cgcaaaacca gcaagaaaag aatgaacaag aattattgga attagataaa tgggcaagtt
     5701 tgtggaattg gtttaacata acaaattggc tgtggtatat aaaattattc ataatgatag
     5761 taggaggctt ggtaggttta agaatagttt ttgctgtact ttctatagtg aatagagtta
     5821 ggcagggata ttcaccatta tcgtttcaga cccacctccc aaccccgagg ggacccgaca
     5881 ggcccgaagg aatagaagaa gaaggtggag agagagacag agacagatcc attcgattag
     5941 tgaacggatc ggcactgcgt gcgccaattc tgcagacaaa tggcagtatt catccacaat
     6001 tttaaaagaa aaggggggat tggggggtac agtgcagggg aaagaatagt agacataata
     6061 gcaacagaca tacaaactaa agaattacaa aaacaaatta caaaaattca aaattttcgg
     6121 gtttattaca gggacagcag agatccagtt tggttaatta actgcagccc cgataaaata
     6181 aaagatttta tttagtctcc agaaaaaggg gggaatgaaa gaccccacct gtaggtttgg
     6241 caagctagct gcagtaacgc cattttgcaa ggcatggaaa aataccaaac caagaataga
     6301 gaagttcaga tcaagggcgg gtacatgaaa atagctaacg ttgggccaaa caggatatct
     6361 gcggtgagca gtttcggccc cggcccgggg ccaagaacag atggtcaccg cagtttcggc
     6421 cccggcccga ggccaagaac agatggtccc cagatatggc ccaaccctca gcagtttctt
     6481 aagacccatc agatgtttcc aggctccccc aaggacctga aatgaccctg cgccttattt
     6541 gaattaacca atcagcctgc ttctcgcttc tgttcgcgcg cttctgcttc ccgagctcta
     6601 taaaagagct cacaacccct cactcggcgc gccagtcctc cgacagactg agtcgcccgg
     6661 gggtctagtg ccaccatgac cgagtacaag cccacggtgc gcctcgccac ccgcgacgac
     6721 gtcccccggg ccgtacgcac cctcgccgcc gcgttcgccg actaccccgc ca
//
