LOCUS       sequence-241998-        7083 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses shRNA targeting the coding sequence of human PHB.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7083)
  AUTHORS   Ortiz MV, Ahmed S, Burns M, Henssen AG, Hollmann TJ, MacArthur I, 
            Gunasekera S, Gaewsky L, Bradwin G, Ryan J, Letai A, He Y, Naranjo 
            A, Chi YY, LaQuaglia M, Heaton T, Cifani P, Dome JS, Gadd S, Perlman
            E, Mullen E, Steen H, Kentsis A
  TITLE     Prohibitin is a prognostic marker and therapeutic target to block 
            chemotherapy resistance in Wilms' tumor.
  JOURNAL   JCI Insight. 2019 Aug 8;4(15). pii: 127098. doi: 
            10.1172/jci.insight.127098. eCollection 2019 Aug 8.
  PUBMED    31391345
REFERENCE   2  (bases 1 to 7083)
  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: "JCI 
            Insight."; date: "2019-08-8"; volume: "4(15). pii: 127098. doi"; 
            pages: " 10.1172/jci.insight.127098. eCollection 2019 Aug 8"
COMMENT     Sequence Label: pLKO-PHB-8
FEATURES             Location/Qualifiers
     source          1..7083
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     40..57
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    174..195
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(210..215,216..233,234..240)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    248..264
                     /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     253..275
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     272..288
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     272..288
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     307..327
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        309..327
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        355..581
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             582..762
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    809..934
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1427..1660
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1845..1889
                     /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        2187..2435
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2187..2207
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2358..2377
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2531..2648
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2697..3207
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     3126..3144
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             3229..3828
                     /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(3229..3248)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3725..3745
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             3956..4189
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4261..4382
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4298..4317)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4352..4371
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4422..4557
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4577..4596)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4578..4596)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4606..4623)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4606..4622)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4615..4637)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4764..5219
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4851..4870)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5061..5082
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5245..5349
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5350..5418,5419..6210)
                     /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(5568..5587)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6381..6969
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6870..6889
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa
       61 gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc
      121 agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg
      181 agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg
      241 tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca tgattacgcc
      301 aagcgcgcaa ttaaccctca ctaaagggaa caaaagctgg agctgcaagc ttaatgtagt
      361 cttatgcaat actcttgtag tcttgcaaca tggtaacgat gagttagcaa catgccttac
      421 aaggagagaa aaagcaccgt gcatgccgat tggtggaagt aaggtggtac gatcgtgcct
      481 tattaggaag gcaacagacg ggtctgacat ggattggacg aaccactgaa ttgccgcatt
      541 gcagagatat tgtatttaag tgcctagctc gatacataaa cgggtctctc tggttagacc
      601 agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag cctcaataaa
      661 gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga
      721 gatccctcag acccttttag tcagtgtgga aaatctctag cagtggcgcc cgaacaggga
      781 cttgaaagcg aaagggaaac cagaggagct ctctcgacgc aggactcggc ttgctgaagc
      841 gcgcacggca agaggcgagg ggcggcgact ggtgagtacg ccaaaaattt tgactagcgg
      901 aggctagaag gagagagatg ggtgcgagag cgtcagtatt aagcggggga gaattagatc
      961 gcgatgggaa aaaattcggt taaggccagg gggaaagaaa aaatataaat taaaacatat
     1021 agtatgggca agcagggagc tagaacgatt cgcagttaat cctggcctgt tagaaacatc
     1081 agaaggctgt agacaaatac tgggacagct acaaccatcc cttcagacag gatcagaaga
     1141 acttagatca ttatataata cagtagcaac cctctattgt gtgcatcaaa ggatagagat
     1201 aaaagacacc aaggaagctt tagacaagat agaggaagag caaaacaaaa gtaagaccac
     1261 cgcacagcaa gcggccgctg atcttcagac ctggaggagg agatatgagg gacaattgga
     1321 gaagtgaatt atataaatat aaagtagtaa aaattgaacc attaggagta gcacccacca
     1381 aggcaaagag aagagtggtg cagagagaaa aaagagcagt gggaatagga gctttgttcc
     1441 ttgggttctt gggagcagca ggaagcacta tgggcgcagc gtcaatgacg ctgacggtac
     1501 aggccagaca attattgtct ggtatagtgc agcagcagaa caatttgctg agggctattg
     1561 aggcgcaaca gcatctgttg caactcacag tctggggcat caagcagctc caggcaagaa
     1621 tcctggctgt ggaaagatac ctaaaggatc aacagctcct ggggatttgg ggttgctctg
     1681 gaaaactcat ttgcaccact gctgtgcctt ggaatgctag ttggagtaat aaatctctgg
     1741 aacagatttg gaatcacacg acctggatgg agtgggacag agaaattaac aattacacaa
     1801 gcttaataca ctccttaatt gaagaatcgc aaaaccagca agaaaagaat gaacaagaat
     1861 tattggaatt agataaatgg gcaagtttgt ggaattggtt taacataaca aattggctgt
     1921 ggtatataaa attattcata atgatagtag gaggcttggt aggtttaaga atagtttttg
     1981 ctgtactttc tatagtgaat agagttaggc agggatattc accattatcg tttcagaccc
     2041 acctcccaac cccgagggga cccgacaggc ccgaaggaat agaagaagaa ggtggagaga
     2101 gagacagaga cagatccatt cgattagtga acggatctcg acggtatcga tcacgagact
     2161 agcctcgagc ggccgccccc ttcaccgagg gcctatttcc catgattcct tcatatttgc
     2221 atatacgata caaggctgtt agagagataa ttggaattaa tttgactgta aacacaaaga
     2281 tattagtaca aaatacgtga cgtagaaagt aataatttct tgggtagttt gcagttttaa
     2341 aattatgttt taaaatggac tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct
     2401 tggctttata tatcttgtgg aaaggacgaa acaccgggag ttcacagaag cggtggaact
     2461 cgagttccac cgcttctgtg aactcttttt aattctcgac ctcgagacaa atggcagtat
     2521 tcatccacaa ttttaaaaga aaagggggga ttggggggta cagtgcaggg gaaagaatag
     2581 tagacataat agcaacagac atacaaacta aagaattaca aaaacaaatt acaaaaattc
     2641 aaaattttcg ggtttattac agggacagca gagatccact ttggccgcgg ctcgaggggg
     2701 ttggggttgc gccttttcca aggcagccct gggtttgcgc agggacgcgg ctgctctggg
     2761 cgtggttccg ggaaacgcag cggcgccgac cctgggtctc gcacattctt cacgtccgtt
     2821 cgcagcgtca cccggatctt cgccgctacc cttgtgggcc ccccggcgac gcttcctgct
     2881 ccgcccctaa gtcgggaagg ttccttgcgg ttcgcggcgt gccggacgtg acaaacggaa
     2941 gccgcacgtc tcactagtac cctcgcagac ggacagcgcc agggagcaat ggcagcgcgc
     3001 cgaccgcgat gggctgtggc caatagcggc tgctcagcag ggcgcgccga gagcagcggc
     3061 cgggaagggg cggtgcggga ggcggggtgt ggggcggtag tgtgggccct gttcctgccc
     3121 gcgcggtgtt ccgcattctg caagcctccg gagcgcacgt cggcagtcgg ctccctcgtt
     3181 gaccgaatca ccgacctctc tccccagggg gatccaccgg agcttaccat gaccgagtac
     3241 aagcccacgg tgcgcctcgc cacccgcgac gacgtcccca gggccgtacg caccctcgcc
     3301 gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg atccggaccg ccacatcgag
     3361 cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg ggctcgacat cggcaaggtg
     3421 tgggtcgcgg acgacggcgc cgcggtggcg gtctggacca cgccggagag cgtcgaagcg
     3481 ggggcggtgt tcgccgagat cggcccgcgc atggccgagt tgagcggttc ccggctggcc
     3541 gcgcagcaac agatggaagg cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc
     3601 ctggccaccg tcggcgtctc gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg
     3661 ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg ccttcctgga gacctccgcg
     3721 ccccgcaacc tccccttcta cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg
     3781 cccgaaggac cgcgcacctg gtgcatgacc cgcaagcccg gtgcctgacg cccgccccac
     3841 gacccgcagc gcccgaccga aaggagcgca cgaccccatg catcggtacc tttaagacca
     3901 atgacttaca aggcagctgt agatcttagc cactttttaa aagaaaaggg gggactggaa
     3961 gggctaattc actcccaacg aagacaagat ctgctttttg cttgtactgg gtctctctgg
     4021 ttagaccaga tctgagcctg ggagctctct ggctaactag ggaacccact gcttaagcct
     4081 caataaagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt
     4141 aactagagat ccctcagacc cttttagtca gtgtggaaaa tctctagcag tagtagttca
     4201 tgtcatctta ttattcagta tttataactt gcaaagaaat gaatatcaga gagtgagagg
     4261 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca
     4321 aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct
     4381 tatcatgtct ggctctagct atcccgcccc taactccgcc catcccgccc ctaactccgc
     4441 ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg
     4501 aggccgcctc ggcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag
     4561 ggacgtaccc aattcgccct atagtgagtc gtattacgcg cgctcactgg ccgtcgtttt
     4621 acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc
     4681 ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt
     4741 gcgcagcctg aatggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     4801 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     4861 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     4921 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
     4981 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     5041 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
     5101 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
     5161 tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaattta
     5221 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     5281 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa
     5341 aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt
     5401 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag
     5461 ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt
     5521 tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg
     5581 gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag
     5641 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta
     5701 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg
     5761 acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta
     5821 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac
     5881 accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt
     5941 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca
     6001 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag
     6061 cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta
     6121 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag
     6181 ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt
     6241 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat
     6301 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     6361 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     6421 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     6481 tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag
     6541 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     6601 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     6661 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     6721 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     6781 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     6841 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     6901 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     6961 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     7021 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt
     7081 gag
//
