LOCUS       sequence-330942-        7084 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral plKO.1 construct coding for a short-hairpin RNA targeting
            the human gene TBK1. 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_shTBK1_3185
FEATURES             Location/Qualifiers
     source          1..7084
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     11..31
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        13..31
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        59..285
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             286..466
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    513..638
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1131..1364
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1549..1593
                     /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        1891..2139
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1891..1911
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2062..2081
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2236..2353
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2402..2912
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     2831..2849
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             2934..3533
                     /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(2934..2953)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3430..3450
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             3661..3894
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    3966..4087
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4003..4022)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4057..4076
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4127..4262
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4282..4301)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4283..4301)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4311..4328)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4311..4327)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4320..4342)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4469..4924
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4556..4575)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4766..4787
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4950..5054
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5055..5123,5124..5915)
                     /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(5273..5292)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6086..6674
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6575..6594
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6828..6845
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6962..6983
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6998..7003,7004..7021,7022..7028)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7036..7052
                     /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     7041..7063
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7060..7076
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7060..7076
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 cgccaagcgc gcaattaacc ctcactaaag ggaacaaaag ctggagctgc aagcttaatg
       61 tagtcttatg caatactctt gtagtcttgc aacatggtaa cgatgagtta gcaacatgcc
      121 ttacaaggag agaaaaagca ccgtgcatgc cgattggtgg aagtaaggtg gtacgatcgt
      181 gccttattag gaaggcaaca gacgggtctg acatggattg gacgaaccac tgaattgccg
      241 cattgcagag atattgtatt taagtgccta gctcgataca taaacgggtc tctctggtta
      301 gaccagatct gagcctggga gctctctggc taactaggga acccactgct taagcctcaa
      361 taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac
      421 tagagatccc tcagaccctt ttagtcagtg tggaaaatct ctagcagtgg cgcccgaaca
      481 gggacttgaa agcgaaaggg aaaccagagg agctctctcg acgcaggact cggcttgctg
      541 aagcgcgcac ggcaagaggc gaggggcggc gactggtgag tacgccaaaa attttgacta
      601 gcggaggcta gaaggagaga gatgggtgcg agagcgtcag tattaagcgg gggagaatta
      661 gatcgcgatg ggaaaaaatt cggttaaggc cagggggaaa gaaaaaatat aaattaaaac
      721 atatagtatg ggcaagcagg gagctagaac gattcgcagt taatcctggc ctgttagaaa
      781 catcagaagg ctgtagacaa atactgggac agctacaacc atcccttcag acaggatcag
      841 aagaacttag atcattatat aatacagtag caaccctcta ttgtgtgcat caaaggatag
      901 agataaaaga caccaaggaa gctttagaca agatagagga agagcaaaac aaaagtaaga
      961 ccaccgcaca gcaagcggcc gctgatcttc agacctggag gaggagatat gagggacaat
     1021 tggagaagtg aattatataa atataaagta gtaaaaattg aaccattagg agtagcaccc
     1081 accaaggcaa agagaagagt ggtgcagaga gaaaaaagag cagtgggaat aggagctttg
     1141 ttccttgggt tcttgggagc agcaggaagc actatgggcg cagcgtcaat gacgctgacg
     1201 gtacaggcca gacaattatt gtctggtata gtgcagcagc agaacaattt gctgagggct
     1261 attgaggcgc aacagcatct gttgcaactc acagtctggg gcatcaagca gctccaggca
     1321 agaatcctgg ctgtggaaag atacctaaag gatcaacagc tcctggggat ttggggttgc
     1381 tctggaaaac tcatttgcac cactgctgtg ccttggaatg ctagttggag taataaatct
     1441 ctggaacaga tttggaatca cacgacctgg atggagtggg acagagaaat taacaattac
     1501 acaagcttaa tacactcctt aattgaagaa tcgcaaaacc agcaagaaaa gaatgaacaa
     1561 gaattattgg aattagataa atgggcaagt ttgtggaatt ggtttaacat aacaaattgg
     1621 ctgtggtata taaaattatt cataatgata gtaggaggct tggtaggttt aagaatagtt
     1681 tttgctgtac tttctatagt gaatagagtt aggcagggat attcaccatt atcgtttcag
     1741 acccacctcc caaccccgag gggacccgac aggcccgaag gaatagaaga agaaggtgga
     1801 gagagagaca gagacagatc cattcgatta gtgaacggat ctcgacggta tcgatcacga
     1861 gactagcctc gagcggccgc ccccttcacc gagggcctat ttcccatgat tccttcatat
     1921 ttgcatatac gatacaaggc tgttagagag ataattggaa ttaatttgac tgtaaacaca
     1981 aagatattag tacaaaatac gtgacgtaga aagtaataat ttcttgggta gtttgcagtt
     2041 ttaaaattat gttttaaaat ggactatcat atgcttaccg taacttgaaa gtatttcgat
     2101 ttcttggctt tatatatctt gtggaaagga cgaaacaccg ggcggcagag ttaggtgaaa
     2161 ttctcgagaa tttcacctaa ctctgccgct ttttgaattc tcgacctcga gacaaatggc
     2221 agtattcatc cacaatttta aaagaaaagg ggggattggg gggtacagtg caggggaaag
     2281 aatagtagac ataatagcaa cagacataca aactaaagaa ttacaaaaac aaattacaaa
     2341 aattcaaaat tttcgggttt attacaggga cagcagagat ccactttggc cgcggctcga
     2401 gggggttggg gttgcgcctt ttccaaggca gccctgggtt tgcgcaggga cgcggctgct
     2461 ctgggcgtgg ttccgggaaa cgcagcggcg ccgaccctgg gtctcgcaca ttcttcacgt
     2521 ccgttcgcag cgtcacccgg atcttcgccg ctacccttgt gggccccccg gcgacgcttc
     2581 ctgctccgcc cctaagtcgg gaaggttcct tgcggttcgc ggcgtgccgg acgtgacaaa
     2641 cggaagccgc acgtctcact agtaccctcg cagacggaca gcgccaggga gcaatggcag
     2701 cgcgccgacc gcgatgggct gtggccaata gcggctgctc agcagggcgc gccgagagca
     2761 gcggccggga aggggcggtg cgggaggcgg ggtgtggggc ggtagtgtgg gccctgttcc
     2821 tgcccgcgcg gtgttccgca ttctgcaagc ctccggagcg cacgtcggca gtcggctccc
     2881 tcgttgaccg aatcaccgac ctctctcccc agggggatcc accggagctt accatgaccg
     2941 agtacaagcc cacggtgcgc ctcgccaccc gcgacgacgt ccccagggcc gtacgcaccc
     3001 tcgccgccgc gttcgccgac taccccgcca cgcgccacac cgtcgatccg gaccgccaca
     3061 tcgagcgggt caccgagctg caagaactct tcctcacgcg cgtcgggctc gacatcggca
     3121 aggtgtgggt cgcggacgac ggcgccgcgg tggcggtctg gaccacgccg gagagcgtcg
     3181 aagcgggggc ggtgttcgcc gagatcggcc cgcgcatggc cgagttgagc ggttcccggc
     3241 tggccgcgca gcaacagatg gaaggcctcc tggcgccgca ccggcccaag gagcccgcgt
     3301 ggttcctggc caccgtcggc gtctcgcccg accaccaggg caagggtctg ggcagcgccg
     3361 tcgtgctccc cggagtggag gcggccgagc gcgccggggt gcccgccttc ctggagacct
     3421 ccgcgccccg caacctcccc ttctacgagc ggctcggctt caccgtcacc gccgacgtcg
     3481 aggtgcccga aggaccgcgc acctggtgca tgacccgcaa gcccggtgcc tgacgcccgc
     3541 cccacgaccc gcagcgcccg accgaaagga gcgcacgacc ccatgcatcg gtacctttaa
     3601 gaccaatgac ttacaaggca gctgtagatc ttagccactt tttaaaagaa aaggggggac
     3661 tggaagggct aattcactcc caacgaagac aagatctgct ttttgcttgt actgggtctc
     3721 tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta
     3781 agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact
     3841 ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagtagta
     3901 gttcatgtca tcttattatt cagtatttat aacttgcaaa gaaatgaata tcagagagtg
     3961 agaggaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt
     4021 tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg
     4081 tatcttatca tgtctggctc tagctatccc gcccctaact ccgcccatcc cgcccctaac
     4141 tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga
     4201 ggccgaggcc gcctcggcct ctgagctatt ccagaagtag tgaggaggct tttttggagg
     4261 cctagggacg tacccaattc gccctatagt gagtcgtatt acgcgcgctc actggccgtc
     4321 gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca
     4381 catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa
     4441 cagttgcgca gcctgaatgg cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg
     4501 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct
     4561 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     4621 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     4681 gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
     4741 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
     4801 cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta
     4861 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca
     4921 atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     4981 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     5041 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     5101 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     5161 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     5221 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     5281 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
     5341 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     5401 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     5461 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     5521 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     5581 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac
     5641 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     5701 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     5761 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     5821 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     5881 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     5941 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     6001 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     6061 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     6121 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     6181 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
     6241 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     6301 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     6361 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     6421 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     6481 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     6541 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     6601 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     6661 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     6721 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
     6781 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
     6841 gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc
     6901 attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa
     6961 ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc
     7021 gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg
     7081 atta
//
