LOCUS       sequence-193106-        7084 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral expression of shRNA targeting Nix.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7084)
  AUTHORS   da Silva Rosa SC, Martens MD, Field JT, Nguyen L, Kereliuk SM, Hai 
            Y, Chapman D, Diehl-Jones W, Aliani M, West AR, Thliveris J, Ghavami
            S, Rampitsch C, Dolinsky VW, Gordon JW
  TITLE     BNIP3L/Nix-induced mitochondrial fission, mitophagy, and impaired 
            myocyte glucose uptake are abrogated by PRKA/PKA phosphorylation.
  JOURNAL   Autophagy. 2021 Sep;17(9):2257-2272. doi: 
            10.1080/15548627.2020.1821548. Epub 2020 Oct 12.
  PUBMED    33044904
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: 
            "10.1080/15548627.2020.1821548"; journalName: "Autophagy"; date: 
            "2021-09"; volume: "17"; issue: "9"; pages: "2257-2272"
COMMENT     Sequence Label: pLKO-sh-Nix
FEATURES             Location/Qualifiers
     source          1..7084
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      20..608
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     509..528
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     762..779
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    896..917
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(932..937,938..955,956..962)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    970..986
                     /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     975..997
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     994..1010
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     994..1010
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     1029..1049
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        1031..1049
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        1077..1303
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             1304..1484
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1531..1656
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    2149..2382
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2567..2611
                     /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        2909..3157
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2909..2929
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     3080..3099
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    3254..3371
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        3420..3930
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     3849..3867
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             3952..4551
                     /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(3952..3971)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4448..4468
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             4679..4912
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4984..5105
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5021..5040)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5075..5094
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      5145..5280
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(5300..5319)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5301..5319)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5329..5346)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5329..5345)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5338..5360)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5487..5942
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5574..5593)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5784..5805
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5968..6072
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6073..6141,6142..6933)
                     /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(6291..6310)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
       61 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
      121 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc
      181 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
      241 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
      301 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
      361 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
      421 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
      481 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
      541 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
      601 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
      661 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
      721 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
      781 aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat
      841 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg
      901 tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt
      961 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg
     1021 ccaagcgcgc aattaaccct cactaaaggg aacaaaagct ggagctgcaa gcttaatgta
     1081 gtcttatgca atactcttgt agtcttgcaa catggtaacg atgagttagc aacatgcctt
     1141 acaaggagag aaaaagcacc gtgcatgccg attggtggaa gtaaggtggt acgatcgtgc
     1201 cttattagga aggcaacaga cgggtctgac atggattgga cgaaccactg aattgccgca
     1261 ttgcagagat attgtattta agtgcctagc tcgatacata aacgggtctc tctggttaga
     1321 ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata
     1381 aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta
     1441 gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg
     1501 gacttgaaag cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa
     1561 gcgcgcacgg caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc
     1621 ggaggctaga aggagagaga tgggtgcgag agcgtcagta ttaagcgggg gagaattaga
     1681 tcgcgatggg aaaaaattcg gttaaggcca gggggaaaga aaaaatataa attaaaacat
     1741 atagtatggg caagcaggga gctagaacga ttcgcagtta atcctggcct gttagaaaca
     1801 tcagaaggct gtagacaaat actgggacag ctacaaccat cccttcagac aggatcagaa
     1861 gaacttagat cattatataa tacagtagca accctctatt gtgtgcatca aaggatagag
     1921 ataaaagaca ccaaggaagc tttagacaag atagaggaag agcaaaacaa aagtaagacc
     1981 accgcacagc aagcggccgc tgatcttcag acctggagga ggagatatga gggacaattg
     2041 gagaagtgaa ttatataaat ataaagtagt aaaaattgaa ccattaggag tagcacccac
     2101 caaggcaaag agaagagtgg tgcagagaga aaaaagagca gtgggaatag gagctttgtt
     2161 ccttgggttc ttgggagcag caggaagcac tatgggcgca gcgtcaatga cgctgacggt
     2221 acaggccaga caattattgt ctggtatagt gcagcagcag aacaatttgc tgagggctat
     2281 tgaggcgcaa cagcatctgt tgcaactcac agtctggggc atcaagcagc tccaggcaag
     2341 aatcctggct gtggaaagat acctaaagga tcaacagctc ctggggattt ggggttgctc
     2401 tggaaaactc atttgcacca ctgctgtgcc ttggaatgct agttggagta ataaatctct
     2461 ggaacagatt tggaatcaca cgacctggat ggagtgggac agagaaatta acaattacac
     2521 aagcttaata cactccttaa ttgaagaatc gcaaaaccag caagaaaaga atgaacaaga
     2581 attattggaa ttagataaat gggcaagttt gtggaattgg tttaacataa caaattggct
     2641 gtggtatata aaattattca taatgatagt aggaggcttg gtaggtttaa gaatagtttt
     2701 tgctgtactt tctatagtga atagagttag gcagggatat tcaccattat cgtttcagac
     2761 ccacctccca accccgaggg gacccgacag gcccgaagga atagaagaag aaggtggaga
     2821 gagagacaga gacagatcca ttcgattagt gaacggatct cgacggtatc gatcacgaga
     2881 ctagcctcga gcggccgccc ccttcaccga gggcctattt cccatgattc cttcatattt
     2941 gcatatacga tacaaggctg ttagagagat aattggaatt aatttgactg taaacacaaa
     3001 gatattagta caaaatacgt gacgtagaaa gtaataattt cttgggtagt ttgcagtttt
     3061 aaaattatgt tttaaaatgg actatcatat gcttaccgta acttgaaagt atttcgattt
     3121 cttggcttta tatatcttgt ggaaaggacg aaacaccgga acagttcctg ggtggagcta
     3181 ctcgagtagc tccacccagg aactgttttt ttgaattctc gacctcgaga caaatggcag
     3241 tattcatcca caattttaaa agaaaagggg ggattggggg gtacagtgca ggggaaagaa
     3301 tagtagacat aatagcaaca gacatacaaa ctaaagaatt acaaaaacaa attacaaaaa
     3361 ttcaaaattt tcgggtttat tacagggaca gcagagatcc actttggccg cggctcgagg
     3421 gggttggggt tgcgcctttt ccaaggcagc cctgggtttg cgcagggacg cggctgctct
     3481 gggcgtggtt ccgggaaacg cagcggcgcc gaccctgggt ctcgcacatt cttcacgtcc
     3541 gttcgcagcg tcacccggat cttcgccgct acccttgtgg gccccccggc gacgcttcct
     3601 gctccgcccc taagtcggga aggttccttg cggttcgcgg cgtgccggac gtgacaaacg
     3661 gaagccgcac gtctcactag taccctcgca gacggacagc gccagggagc aatggcagcg
     3721 cgccgaccgc gatgggctgt ggccaatagc ggctgctcag cagggcgcgc cgagagcagc
     3781 ggccgggaag gggcggtgcg ggaggcgggg tgtggggcgg tagtgtgggc cctgttcctg
     3841 cccgcgcggt gttccgcatt ctgcaagcct ccggagcgca cgtcggcagt cggctccctc
     3901 gttgaccgaa tcaccgacct ctctccccag ggggatccac cggagcttac catgaccgag
     3961 tacaagccca cggtgcgcct cgccacccgc gacgacgtcc ccagggccgt acgcaccctc
     4021 gccgccgcgt tcgccgacta ccccgccacg cgccacaccg tcgatccgga ccgccacatc
     4081 gagcgggtca ccgagctgca agaactcttc ctcacgcgcg tcgggctcga catcggcaag
     4141 gtgtgggtcg cggacgacgg cgccgcggtg gcggtctgga ccacgccgga gagcgtcgaa
     4201 gcgggggcgg tgttcgccga gatcggcccg cgcatggccg agttgagcgg ttcccggctg
     4261 gccgcgcagc aacagatgga aggcctcctg gcgccgcacc ggcccaagga gcccgcgtgg
     4321 ttcctggcca ccgtcggcgt ctcgcccgac caccagggca agggtctggg cagcgccgtc
     4381 gtgctccccg gagtggaggc ggccgagcgc gccggggtgc ccgccttcct ggagacctcc
     4441 gcgccccgca acctcccctt ctacgagcgg ctcggcttca ccgtcaccgc cgacgtcgag
     4501 gtgcccgaag gaccgcgcac ctggtgcatg acccgcaagc ccggtgcctg acgcccgccc
     4561 cacgacccgc agcgcccgac cgaaaggagc gcacgacccc atgcatcggt acctttaaga
     4621 ccaatgactt acaaggcagc tgtagatctt agccactttt taaaagaaaa ggggggactg
     4681 gaagggctaa ttcactccca acgaagacaa gatctgcttt ttgcttgtac tgggtctctc
     4741 tggttagacc agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag
     4801 cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct
     4861 ggtaactaga gatccctcag acccttttag tcagtgtgga aaatctctag cagtagtagt
     4921 tcatgtcatc ttattattca gtatttataa cttgcaaaga aatgaatatc agagagtgag
     4981 aggaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc
     5041 acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta
     5101 tcttatcatg tctggctcta gctatcccgc ccctaactcc gcccatcccg cccctaactc
     5161 cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg
     5221 ccgaggccgc ctcggcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc
     5281 tagggacgta cccaattcgc cctatagtga gtcgtattac gcgcgctcac tggccgtcgt
     5341 tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca
     5401 tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca
     5461 gttgcgcagc ctgaatggcg aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg
     5521 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
     5581 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     5641 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     5701 ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
     5761 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc
     5821 tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa
     5881 aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat
     5941 ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     6001 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
     6061 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca
     6121 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat
     6181 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag
     6241 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc
     6301 gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct
     6361 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca
     6421 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt
     6481 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat
     6541 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt
     6601 gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta
     6661 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga
     6721 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt
     6781 gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc
     6841 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct
     6901 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata
     6961 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
     7021 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     7081 gtag
//
