LOCUS       sequence-330944-        7084 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral plKO.1 construct coding for a short-hairpin RNA targeting
            the human gene DDX58 (coding for RLR sensor RIG-I). Contains a puro 
            resistance cassette..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7084)
  AUTHORS   Tigano M, Vargas DC, Tremblay-Belzile S, Fu Y, Sfeir A
  TITLE     Nuclear sensing of breaks in mitochondrial DNA enhances immune 
            surveillance.
  JOURNAL   Nature. 2021 Mar;591(7850):477-481. doi: 10.1038/s41586-021-03269-w.
            Epub 2021 Feb 24.
  PUBMED    33627873
REFERENCE   2  (bases 1 to 7084)
  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"; doi: 
            "10.1038/s41586-021-03269-w"; journalName: "Nature"; date: 
            "2021-03"; volume: "591"; issue: "7850"; pages: "477-481"
COMMENT     Sequence Label: pLKO.1 puro_shDDX58_230212
FEATURES             Location/Qualifiers
     source          1..7084
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     8..28
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        10..28
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        56..282
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             283..463
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    510..635
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1128..1361
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1546..1590
                     /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"
     promoter        1888..2136
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1888..1908
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2059..2078
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2233..2350
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2399..2909
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     2828..2846
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             2931..3530
                     /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(2931..2950)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3427..3447
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             3658..3891
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    3963..4084
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4000..4019)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4054..4073
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4124..4259
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4279..4298)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4280..4298)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4308..4325)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4308..4324)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4317..4339)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4466..4921
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4553..4572)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4763..4784
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4947..5051
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5052..5120,5121..5912)
                     /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     complement(5270..5289)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6083..6671
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6572..6591
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6825..6842
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6959..6980
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6995..7000,7001..7018,7019..7025)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7033..7049
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     7038..7060
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7057..7073
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7057..7073
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 caagcgcgca attaaccctc actaaaggga acaaaagctg gagctgcaag cttaatgtag
       61 tcttatgcaa tactcttgta gtcttgcaac atggtaacga tgagttagca acatgcctta
      121 caaggagaga aaaagcaccg tgcatgccga ttggtggaag taaggtggta cgatcgtgcc
      181 ttattaggaa ggcaacagac gggtctgaca tggattggac gaaccactga attgccgcat
      241 tgcagagata ttgtatttaa gtgcctagct cgatacataa acgggtctct ctggttagac
      301 cagatctgag cctgggagct ctctggctaa ctagggaacc cactgcttaa gcctcaataa
      361 agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag
      421 agatccctca gaccctttta gtcagtgtgg aaaatctcta gcagtggcgc ccgaacaggg
      481 acttgaaagc gaaagggaaa ccagaggagc tctctcgacg caggactcgg cttgctgaag
      541 cgcgcacggc aagaggcgag gggcggcgac tggtgagtac gccaaaaatt ttgactagcg
      601 gaggctagaa ggagagagat gggtgcgaga gcgtcagtat taagcggggg agaattagat
      661 cgcgatggga aaaaattcgg ttaaggccag ggggaaagaa aaaatataaa ttaaaacata
      721 tagtatgggc aagcagggag ctagaacgat tcgcagttaa tcctggcctg ttagaaacat
      781 cagaaggctg tagacaaata ctgggacagc tacaaccatc ccttcagaca ggatcagaag
      841 aacttagatc attatataat acagtagcaa ccctctattg tgtgcatcaa aggatagaga
      901 taaaagacac caaggaagct ttagacaaga tagaggaaga gcaaaacaaa agtaagacca
      961 ccgcacagca agcggccgct gatcttcaga cctggaggag gagatatgag ggacaattgg
     1021 agaagtgaat tatataaata taaagtagta aaaattgaac cattaggagt agcacccacc
     1081 aaggcaaaga gaagagtggt gcagagagaa aaaagagcag tgggaatagg agctttgttc
     1141 cttgggttct tgggagcagc aggaagcact atgggcgcag cgtcaatgac gctgacggta
     1201 caggccagac aattattgtc tggtatagtg cagcagcaga acaatttgct gagggctatt
     1261 gaggcgcaac agcatctgtt gcaactcaca gtctggggca tcaagcagct ccaggcaaga
     1321 atcctggctg tggaaagata cctaaaggat caacagctcc tggggatttg gggttgctct
     1381 ggaaaactca tttgcaccac tgctgtgcct tggaatgcta gttggagtaa taaatctctg
     1441 gaacagattt ggaatcacac gacctggatg gagtgggaca gagaaattaa caattacaca
     1501 agcttaatac actccttaat tgaagaatcg caaaaccagc aagaaaagaa tgaacaagaa
     1561 ttattggaat tagataaatg ggcaagtttg tggaattggt ttaacataac aaattggctg
     1621 tggtatataa aattattcat aatgatagta ggaggcttgg taggtttaag aatagttttt
     1681 gctgtacttt ctatagtgaa tagagttagg cagggatatt caccattatc gtttcagacc
     1741 cacctcccaa ccccgagggg acccgacagg cccgaaggaa tagaagaaga aggtggagag
     1801 agagacagag acagatccat tcgattagtg aacggatctc gacggtatcg atcacgagac
     1861 tagcctcgag cggccgcccc cttcaccgag ggcctatttc ccatgattcc ttcatatttg
     1921 catatacgat acaaggctgt tagagagata attggaatta atttgactgt aaacacaaag
     1981 atattagtac aaaatacgtg acgtagaaag taataatttc ttgggtagtt tgcagtttta
     2041 aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta tttcgatttc
     2101 ttggctttat atatcttgtg gaaaggacga aacaccggag cacttgtgga cgctttaaac
     2161 tcgagtttaa agcgtccaca agtgcttttt tgaattctcg acctcgagac aaatggcagt
     2221 attcatccac aattttaaaa gaaaaggggg gattgggggg tacagtgcag gggaaagaat
     2281 agtagacata atagcaacag acatacaaac taaagaatta caaaaacaaa ttacaaaaat
     2341 tcaaaatttt cgggtttatt acagggacag cagagatcca ctttggccgc ggctcgaggg
     2401 ggttggggtt gcgccttttc caaggcagcc ctgggtttgc gcagggacgc ggctgctctg
     2461 ggcgtggttc cgggaaacgc agcggcgccg accctgggtc tcgcacattc ttcacgtccg
     2521 ttcgcagcgt cacccggatc ttcgccgcta cccttgtggg ccccccggcg acgcttcctg
     2581 ctccgcccct aagtcgggaa ggttccttgc ggttcgcggc gtgccggacg tgacaaacgg
     2641 aagccgcacg tctcactagt accctcgcag acggacagcg ccagggagca atggcagcgc
     2701 gccgaccgcg atgggctgtg gccaatagcg gctgctcagc agggcgcgcc gagagcagcg
     2761 gccgggaagg ggcggtgcgg gaggcggggt gtggggcggt agtgtgggcc ctgttcctgc
     2821 ccgcgcggtg ttccgcattc tgcaagcctc cggagcgcac gtcggcagtc ggctccctcg
     2881 ttgaccgaat caccgacctc tctccccagg gggatccacc ggagcttacc atgaccgagt
     2941 acaagcccac ggtgcgcctc gccacccgcg acgacgtccc cagggccgta cgcaccctcg
     3001 ccgccgcgtt cgccgactac cccgccacgc gccacaccgt cgatccggac cgccacatcg
     3061 agcgggtcac cgagctgcaa gaactcttcc tcacgcgcgt cgggctcgac atcggcaagg
     3121 tgtgggtcgc ggacgacggc gccgcggtgg cggtctggac cacgccggag agcgtcgaag
     3181 cgggggcggt gttcgccgag atcggcccgc gcatggccga gttgagcggt tcccggctgg
     3241 ccgcgcagca acagatggaa ggcctcctgg cgccgcaccg gcccaaggag cccgcgtggt
     3301 tcctggccac cgtcggcgtc tcgcccgacc accagggcaa gggtctgggc agcgccgtcg
     3361 tgctccccgg agtggaggcg gccgagcgcg ccggggtgcc cgccttcctg gagacctccg
     3421 cgccccgcaa cctccccttc tacgagcggc tcggcttcac cgtcaccgcc gacgtcgagg
     3481 tgcccgaagg accgcgcacc tggtgcatga cccgcaagcc cggtgcctga cgcccgcccc
     3541 acgacccgca gcgcccgacc gaaaggagcg cacgacccca tgcatcggta cctttaagac
     3601 caatgactta caaggcagct gtagatctta gccacttttt aaaagaaaag gggggactgg
     3661 aagggctaat tcactcccaa cgaagacaag atctgctttt tgcttgtact gggtctctct
     3721 ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca ctgcttaagc
     3781 ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg
     3841 gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc agtagtagtt
     3901 catgtcatct tattattcag tatttataac ttgcaaagaa atgaatatca gagagtgaga
     3961 ggaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
     4021 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat
     4081 cttatcatgt ctggctctag ctatcccgcc cctaactccg cccatcccgc ccctaactcc
     4141 gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc
     4201 cgaggccgcc tcggcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct
     4261 agggacgtac ccaattcgcc ctatagtgag tcgtattacg cgcgctcact ggccgtcgtt
     4321 ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat
     4381 ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag
     4441 ttgcgcagcc tgaatggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt
     4501 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc
     4561 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
     4621 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
     4681 tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
     4741 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct
     4801 atctcggtct attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa
     4861 aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt
     4921 taggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     4981 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     5041 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
     5101 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
     5161 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
     5221 gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg
     5281 cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc
     5341 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
     5401 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
     5461 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
     5521 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
     5581 acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
     5641 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
     5701 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
     5761 agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg
     5821 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
     5881 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac
     5941 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
     6001 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     6061 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     6121 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     6181 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt
     6241 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     6301 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     6361 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     6421 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     6481 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     6541 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     6601 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     6661 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     6721 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct
     6781 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg
     6841 aggaagcgga agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt
     6901 aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta
     6961 atgtgagtta gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta
     7021 tgttgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt
     7081 acgc
//
