LOCUS       sequence-241997-        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-6
FEATURES             Location/Qualifiers
     source          1..7083
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    62..83
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(98..103,104..121,122..128)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    136..152
                     /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     141..163
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     160..176
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     160..176
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     195..215
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        197..215
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        243..469
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             470..650
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    697..822
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1315..1548
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1733..1777
                     /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        2075..2323
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2075..2095
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2246..2265
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2419..2536
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2585..3095
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     3014..3032
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             3117..3716
                     /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(3117..3136)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3613..3633
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             3844..4077
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4149..4270
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4186..4205)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4240..4259
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4310..4445
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4465..4484)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4466..4484)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4494..4511)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4494..4510)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4503..4525)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4652..5107
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4739..4758)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4949..4970
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5133..5237
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5238..5306,5307..6098)
                     /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(5456..5475)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6269..6857
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6758..6777
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7011..7028
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa
       61 ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc
      121 gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg
      181 attacgccaa gcgcgcaatt aaccctcact aaagggaaca aaagctggag ctgcaagctt
      241 aatgtagtct tatgcaatac tcttgtagtc ttgcaacatg gtaacgatga gttagcaaca
      301 tgccttacaa ggagagaaaa agcaccgtgc atgccgattg gtggaagtaa ggtggtacga
      361 tcgtgcctta ttaggaaggc aacagacggg tctgacatgg attggacgaa ccactgaatt
      421 gccgcattgc agagatattg tatttaagtg cctagctcga tacataaacg ggtctctctg
      481 gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc
      541 tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg
      601 taactagaga tccctcagac ccttttagtc agtgtggaaa atctctagca gtggcgcccg
      661 aacagggact tgaaagcgaa agggaaacca gaggagctct ctcgacgcag gactcggctt
      721 gctgaagcgc gcacggcaag aggcgagggg cggcgactgg tgagtacgcc aaaaattttg
      781 actagcggag gctagaagga gagagatggg tgcgagagcg tcagtattaa gcgggggaga
      841 attagatcgc gatgggaaaa aattcggtta aggccagggg gaaagaaaaa atataaatta
      901 aaacatatag tatgggcaag cagggagcta gaacgattcg cagttaatcc tggcctgtta
      961 gaaacatcag aaggctgtag acaaatactg ggacagctac aaccatccct tcagacagga
     1021 tcagaagaac ttagatcatt atataataca gtagcaaccc tctattgtgt gcatcaaagg
     1081 atagagataa aagacaccaa ggaagcttta gacaagatag aggaagagca aaacaaaagt
     1141 aagaccaccg cacagcaagc ggccgctgat cttcagacct ggaggaggag atatgaggga
     1201 caattggaga agtgaattat ataaatataa agtagtaaaa attgaaccat taggagtagc
     1261 acccaccaag gcaaagagaa gagtggtgca gagagaaaaa agagcagtgg gaataggagc
     1321 tttgttcctt gggttcttgg gagcagcagg aagcactatg ggcgcagcgt caatgacgct
     1381 gacggtacag gccagacaat tattgtctgg tatagtgcag cagcagaaca atttgctgag
     1441 ggctattgag gcgcaacagc atctgttgca actcacagtc tggggcatca agcagctcca
     1501 ggcaagaatc ctggctgtgg aaagatacct aaaggatcaa cagctcctgg ggatttgggg
     1561 ttgctctgga aaactcattt gcaccactgc tgtgccttgg aatgctagtt ggagtaataa
     1621 atctctggaa cagatttgga atcacacgac ctggatggag tgggacagag aaattaacaa
     1681 ttacacaagc ttaatacact ccttaattga agaatcgcaa aaccagcaag aaaagaatga
     1741 acaagaatta ttggaattag ataaatgggc aagtttgtgg aattggttta acataacaaa
     1801 ttggctgtgg tatataaaat tattcataat gatagtagga ggcttggtag gtttaagaat
     1861 agtttttgct gtactttcta tagtgaatag agttaggcag ggatattcac cattatcgtt
     1921 tcagacccac ctcccaaccc cgaggggacc cgacaggccc gaaggaatag aagaagaagg
     1981 tggagagaga gacagagaca gatccattcg attagtgaac ggatctcgac ggtatcgatc
     2041 acgagactag cctcgagcgg ccgccccctt caccgagggc ctatttccca tgattccttc
     2101 atatttgcat atacgataca aggctgttag agagataatt ggaattaatt tgactgtaaa
     2161 cacaaagata ttagtacaaa atacgtgacg tagaaagtaa taatttcttg ggtagtttgc
     2221 agttttaaaa ttatgtttta aaatggacta tcatatgctt accgtaactt gaaagtattt
     2281 cgatttcttg gctttatata tcttgtggaa aggacgaaac accggcgtgg tgaactctgc
     2341 cttatactcg agtataaggc agagttcacc acgtttttaa ttctcgacct cgagacaaat
     2401 ggcagtattc atccacaatt ttaaaagaaa aggggggatt ggggggtaca gtgcagggga
     2461 aagaatagta gacataatag caacagacat acaaactaaa gaattacaaa aacaaattac
     2521 aaaaattcaa aattttcggg tttattacag ggacagcaga gatccacttt ggccgcggct
     2581 cgagggggtt ggggttgcgc cttttccaag gcagccctgg gtttgcgcag ggacgcggct
     2641 gctctgggcg tggttccggg aaacgcagcg gcgccgaccc tgggtctcgc acattcttca
     2701 cgtccgttcg cagcgtcacc cggatcttcg ccgctaccct tgtgggcccc ccggcgacgc
     2761 ttcctgctcc gcccctaagt cgggaaggtt ccttgcggtt cgcggcgtgc cggacgtgac
     2821 aaacggaagc cgcacgtctc actagtaccc tcgcagacgg acagcgccag ggagcaatgg
     2881 cagcgcgccg accgcgatgg gctgtggcca atagcggctg ctcagcaggg cgcgccgaga
     2941 gcagcggccg ggaaggggcg gtgcgggagg cggggtgtgg ggcggtagtg tgggccctgt
     3001 tcctgcccgc gcggtgttcc gcattctgca agcctccgga gcgcacgtcg gcagtcggct
     3061 ccctcgttga ccgaatcacc gacctctctc cccaggggga tccaccggag cttaccatga
     3121 ccgagtacaa gcccacggtg cgcctcgcca cccgcgacga cgtccccagg gccgtacgca
     3181 ccctcgccgc cgcgttcgcc gactaccccg ccacgcgcca caccgtcgat ccggaccgcc
     3241 acatcgagcg ggtcaccgag ctgcaagaac tcttcctcac gcgcgtcggg ctcgacatcg
     3301 gcaaggtgtg ggtcgcggac gacggcgccg cggtggcggt ctggaccacg ccggagagcg
     3361 tcgaagcggg ggcggtgttc gccgagatcg gcccgcgcat ggccgagttg agcggttccc
     3421 ggctggccgc gcagcaacag atggaaggcc tcctggcgcc gcaccggccc aaggagcccg
     3481 cgtggttcct ggccaccgtc ggcgtctcgc ccgaccacca gggcaagggt ctgggcagcg
     3541 ccgtcgtgct ccccggagtg gaggcggccg agcgcgccgg ggtgcccgcc ttcctggaga
     3601 cctccgcgcc ccgcaacctc cccttctacg agcggctcgg cttcaccgtc accgccgacg
     3661 tcgaggtgcc cgaaggaccg cgcacctggt gcatgacccg caagcccggt gcctgacgcc
     3721 cgccccacga cccgcagcgc ccgaccgaaa ggagcgcacg accccatgca tcggtacctt
     3781 taagaccaat gacttacaag gcagctgtag atcttagcca ctttttaaaa gaaaaggggg
     3841 gactggaagg gctaattcac tcccaacgaa gacaagatct gctttttgct tgtactgggt
     3901 ctctctggtt agaccagatc tgagcctggg agctctctgg ctaactaggg aacccactgc
     3961 ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg
     4021 actctggtaa ctagagatcc ctcagaccct tttagtcagt gtggaaaatc tctagcagta
     4081 gtagttcatg tcatcttatt attcagtatt tataacttgc aaagaaatga atatcagaga
     4141 gtgagaggaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
     4201 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
     4261 atgtatctta tcatgtctgg ctctagctat cccgccccta actccgccca tcccgcccct
     4321 aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc
     4381 agaggccgag gccgcctcgg cctctgagct attccagaag tagtgaggag gcttttttgg
     4441 aggcctaggg acgtacccaa ttcgccctat agtgagtcgt attacgcgcg ctcactggcc
     4501 gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa tcgccttgca
     4561 gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc
     4621 caacagttgc gcagcctgaa tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg
     4681 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct
     4741 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
     4801 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
     4861 cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
     4921 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
     4981 aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg
     5041 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgctt
     5101 acaatttagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     5161 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
     5221 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
     5281 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
     5341 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
     5401 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat
     5461 gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact
     5521 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
     5581 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
     5641 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
     5701 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
     5761 agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg
     5821 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
     5881 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
     5941 ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc
     6001 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
     6061 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
     6121 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
     6181 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     6241 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     6301 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     6361 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc
     6421 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     6481 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     6541 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     6601 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     6661 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     6721 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     6781 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     6841 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     6901 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
     6961 ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag
     7021 tgagcgagga agcggaagag cgcccaatac gcaaaccgcc tctccccgcg cgttggccga
     7081 ttc
//
