LOCUS       sequence-242000-        7083 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses shRNA targeting the 3' UTR 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-4
FEATURES             Location/Qualifiers
     source          1..7083
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     90..107
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    224..245
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(260..265,266..283,284..290)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    298..314
                     /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     303..325
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     322..338
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     322..338
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     357..377
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        359..377
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        405..631
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             632..812
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    859..984
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1477..1710
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1895..1939
                     /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        2237..2485
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2237..2257
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2408..2427
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2581..2698
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2747..3257
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     3176..3194
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             3279..3878
                     /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(3279..3298)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3775..3795
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             4006..4239
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4311..4432
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4348..4367)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4402..4421
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4472..4607
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4627..4646)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4628..4646)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4656..4673)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4656..4672)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4665..4687)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4814..5269
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4901..4920)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5111..5132
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5295..5399
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5400..5468,5469..6260)
                     /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(5618..5637)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6431..7019
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6920..6939
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata
       61 ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc
      121 gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg
      181 acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg agttagctca
      241 ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg tgtggaattg
      301 tgagcggata acaatttcac acaggaaaca gctatgacca tgattacgcc aagcgcgcaa
      361 ttaaccctca ctaaagggaa caaaagctgg agctgcaagc ttaatgtagt cttatgcaat
      421 actcttgtag tcttgcaaca tggtaacgat gagttagcaa catgccttac aaggagagaa
      481 aaagcaccgt gcatgccgat tggtggaagt aaggtggtac gatcgtgcct tattaggaag
      541 gcaacagacg ggtctgacat ggattggacg aaccactgaa ttgccgcatt gcagagatat
      601 tgtatttaag tgcctagctc gatacataaa cgggtctctc tggttagacc agatctgagc
      661 ctgggagctc tctggctaac tagggaaccc actgcttaag cctcaataaa gcttgccttg
      721 agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga gatccctcag
      781 acccttttag tcagtgtgga aaatctctag cagtggcgcc cgaacaggga cttgaaagcg
      841 aaagggaaac cagaggagct ctctcgacgc aggactcggc ttgctgaagc gcgcacggca
      901 agaggcgagg ggcggcgact ggtgagtacg ccaaaaattt tgactagcgg aggctagaag
      961 gagagagatg ggtgcgagag cgtcagtatt aagcggggga gaattagatc gcgatgggaa
     1021 aaaattcggt taaggccagg gggaaagaaa aaatataaat taaaacatat agtatgggca
     1081 agcagggagc tagaacgatt cgcagttaat cctggcctgt tagaaacatc agaaggctgt
     1141 agacaaatac tgggacagct acaaccatcc cttcagacag gatcagaaga acttagatca
     1201 ttatataata cagtagcaac cctctattgt gtgcatcaaa ggatagagat aaaagacacc
     1261 aaggaagctt tagacaagat agaggaagag caaaacaaaa gtaagaccac cgcacagcaa
     1321 gcggccgctg atcttcagac ctggaggagg agatatgagg gacaattgga gaagtgaatt
     1381 atataaatat aaagtagtaa aaattgaacc attaggagta gcacccacca aggcaaagag
     1441 aagagtggtg cagagagaaa aaagagcagt gggaatagga gctttgttcc ttgggttctt
     1501 gggagcagca ggaagcacta tgggcgcagc gtcaatgacg ctgacggtac aggccagaca
     1561 attattgtct ggtatagtgc agcagcagaa caatttgctg agggctattg aggcgcaaca
     1621 gcatctgttg caactcacag tctggggcat caagcagctc caggcaagaa tcctggctgt
     1681 ggaaagatac ctaaaggatc aacagctcct ggggatttgg ggttgctctg gaaaactcat
     1741 ttgcaccact gctgtgcctt ggaatgctag ttggagtaat aaatctctgg aacagatttg
     1801 gaatcacacg acctggatgg agtgggacag agaaattaac aattacacaa gcttaataca
     1861 ctccttaatt gaagaatcgc aaaaccagca agaaaagaat gaacaagaat tattggaatt
     1921 agataaatgg gcaagtttgt ggaattggtt taacataaca aattggctgt ggtatataaa
     1981 attattcata atgatagtag gaggcttggt aggtttaaga atagtttttg ctgtactttc
     2041 tatagtgaat agagttaggc agggatattc accattatcg tttcagaccc acctcccaac
     2101 cccgagggga cccgacaggc ccgaaggaat agaagaagaa ggtggagaga gagacagaga
     2161 cagatccatt cgattagtga acggatctcg acggtatcga tcacgagact agcctcgagc
     2221 ggccgccccc ttcaccgagg gcctatttcc catgattcct tcatatttgc atatacgata
     2281 caaggctgtt agagagataa ttggaattaa tttgactgta aacacaaaga tattagtaca
     2341 aaatacgtga cgtagaaagt aataatttct tgggtagttt gcagttttaa aattatgttt
     2401 taaaatggac tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct tggctttata
     2461 tatcttgtgg aaaggacgaa acaccggccc agaaatcact gtgaaattct cgagaatttc
     2521 acagtgattt ctgggttttt aattctcgac ctcgagacaa atggcagtat tcatccacaa
     2581 ttttaaaaga aaagggggga ttggggggta cagtgcaggg gaaagaatag tagacataat
     2641 agcaacagac atacaaacta aagaattaca aaaacaaatt acaaaaattc aaaattttcg
     2701 ggtttattac agggacagca gagatccact ttggccgcgg ctcgaggggg ttggggttgc
     2761 gccttttcca aggcagccct gggtttgcgc agggacgcgg ctgctctggg cgtggttccg
     2821 ggaaacgcag cggcgccgac cctgggtctc gcacattctt cacgtccgtt cgcagcgtca
     2881 cccggatctt cgccgctacc cttgtgggcc ccccggcgac gcttcctgct ccgcccctaa
     2941 gtcgggaagg ttccttgcgg ttcgcggcgt gccggacgtg acaaacggaa gccgcacgtc
     3001 tcactagtac cctcgcagac ggacagcgcc agggagcaat ggcagcgcgc cgaccgcgat
     3061 gggctgtggc caatagcggc tgctcagcag ggcgcgccga gagcagcggc cgggaagggg
     3121 cggtgcggga ggcggggtgt ggggcggtag tgtgggccct gttcctgccc gcgcggtgtt
     3181 ccgcattctg caagcctccg gagcgcacgt cggcagtcgg ctccctcgtt gaccgaatca
     3241 ccgacctctc tccccagggg gatccaccgg agcttaccat gaccgagtac aagcccacgg
     3301 tgcgcctcgc cacccgcgac gacgtcccca gggccgtacg caccctcgcc gccgcgttcg
     3361 ccgactaccc cgccacgcgc cacaccgtcg atccggaccg ccacatcgag cgggtcaccg
     3421 agctgcaaga actcttcctc acgcgcgtcg ggctcgacat cggcaaggtg tgggtcgcgg
     3481 acgacggcgc cgcggtggcg gtctggacca cgccggagag cgtcgaagcg ggggcggtgt
     3541 tcgccgagat cggcccgcgc atggccgagt tgagcggttc ccggctggcc gcgcagcaac
     3601 agatggaagg cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc ctggccaccg
     3661 tcggcgtctc gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg ctccccggag
     3721 tggaggcggc cgagcgcgcc ggggtgcccg ccttcctgga gacctccgcg ccccgcaacc
     3781 tccccttcta cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg cccgaaggac
     3841 cgcgcacctg gtgcatgacc cgcaagcccg gtgcctgacg cccgccccac gacccgcagc
     3901 gcccgaccga aaggagcgca cgaccccatg catcggtacc tttaagacca atgacttaca
     3961 aggcagctgt agatcttagc cactttttaa aagaaaaggg gggactggaa gggctaattc
     4021 actcccaacg aagacaagat ctgctttttg cttgtactgg gtctctctgg ttagaccaga
     4081 tctgagcctg ggagctctct ggctaactag ggaacccact gcttaagcct caataaagct
     4141 tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt aactagagat
     4201 ccctcagacc cttttagtca gtgtggaaaa tctctagcag tagtagttca tgtcatctta
     4261 ttattcagta tttataactt gcaaagaaat gaatatcaga gagtgagagg aacttgttta
     4321 ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat
     4381 ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct
     4441 ggctctagct atcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc
     4501 ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc
     4561 ggcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag ggacgtaccc
     4621 aattcgccct atagtgagtc gtattacgcg cgctcactgg ccgtcgtttt acaacgtcgt
     4681 gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc
     4741 agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg
     4801 aatggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     4861 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     4921 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     4981 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt
     5041 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     5101 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat
     5161 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
     5221 taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaattta ggtggcactt
     5281 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     5341 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta
     5401 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
     5461 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac
     5521 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg
     5581 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc
     5641 gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg
     5701 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat
     5761 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg
     5821 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg
     5881 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc
     5941 ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt
     6001 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct
     6061 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc
     6121 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca
     6181 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct
     6241 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt
     6301 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga
     6361 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     6421 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     6481 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     6541 taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag
     6601 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     6661 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     6721 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     6781 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     6841 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     6901 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     6961 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     7021 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     7081 tct
//
