LOCUS       sequence-306297-        6371 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a shRNA against firefly luciferase.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6371)
  AUTHORS   Hyodo T, Rahman ML, Karnan S, Ito T, Toyoda A, Ota A, Wahiduzzaman 
            M, Tsuzuki S, Okada Y, Hosokawa Y, Konishi H
  TITLE     Tandem Paired Nicking Promotes Precise Genome Editing with Scarce 
            Interference by p53.
  JOURNAL   Cell Rep. 2020 Jan 28;30(4):1195-1207.e7. doi: 
            10.1016/j.celrep.2019.12.064.
  PUBMED    31995758
REFERENCE   2  (bases 1 to 6371)
  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: 
            "10.1016/j.celrep.2019.12"; journalName: "Cell Rep"; date: 
            "2020-01-28- 28"; volume: "30"; issue: "4"; pages: "1195-1207.e7"
COMMENT     Sequence Label: pMKO.1 puro-luc (shRNA)
FEATURES             Location/Qualifiers
     source          1..6371
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        19..267
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     19..39
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     190..209
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     promoter        359..688
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      539..674
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             698..1297
                     /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(698..717)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1194..1214
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             1493..1918
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     Moloney murine leukemia virus"
     primer_bind     complement(2021..2043)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     promoter        2184..2513
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2364..2499
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2535..2554
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(2635..2652)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2806..3394)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2886..2905)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3565..4356,4357..4425))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     4188..4207
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4426..4530)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4598..4616
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     enhancer        4716..5095
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        5096..5299
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     5250..5270
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             5300..5475
                     /label=5' LTR (truncated)
                     /note="truncated long terminal repeat from Moloney murine 
                     sarcoma virus"
     misc_feature    5538..5737
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             5938..6354
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     6284..6306
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     6322..6338
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
ORIGIN
        1 gacaaggtcg ggcaggaaga gggcctattt cccatgattc cttcatattt gcatatacga
       61 tacaaggctg ttagagagat aattagaatt aatttgactg taaacacaaa gatattagta
      121 caaaatacgt gacgtagaaa gtaataattt cttgggtagt ttgcagtttt aaaattatgt
      181 tttaaaatgg actatcatat gcttaccgta acttgaaagt atttcgattt cttggcttta
      241 tatatcttgt ggaaaggacg aaacaccggt gcttacgctg agtacttcga ttcaagagat
      301 cgaagtactc agcgtaagtt ttttgaattc gccagcacag tggtcgaccc tgtggaatgt
      361 gtgtcagtta gggtgtggaa agtccccagg cttcccagca ggcagaagta tgcaaagcat
      421 gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag
      481 tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat
      541 cccgccccta actccgccca gttccgccca ttctccgccc catggctgac taattttttt
      601 tatttatgca gaggccgagg ccgcctcggc ctctgagcta ttccagaagt agtgaggagg
      661 cttttttgga ggcctaggct tttgcaaaaa gcttaccatg accgagtaca agcccacggt
      721 gcgcctcgcc acccgcgacg acgtccccag ggccgtacgc accctcgccg ccgcgttcgc
      781 cgactacccc gccacgcgcc acaccgtcga tccggaccgc cacatcgagc gggtcaccga
      841 gctgcaagaa ctcttcctca cgcgcgtcgg gctcgacatc ggcaaggtgt gggtcgcgga
      901 cgacggcgcc gcggtggcgg tctggaccac gccggagagc gtcgaagcgg gggcggtgtt
      961 cgccgagatc ggcccgcgca tggccgagtt gagcggttcc cggctggccg cgcagcaaca
     1021 gatggaaggc ctcctggcgc cgcaccggcc caaggagccc gcgtggttcc tggccaccgt
     1081 cggcgtctcg cccgaccacc agggcaaggg tctgggcagc gccgtcgtgc tccccggagt
     1141 ggaggcggcc gagcgcgccg gggtgcccgc cttcctggag acctccgcgc cccgcaacct
     1201 ccccttctac gagcggctcg gcttcaccgt caccgccgac gtcgaggtgc ccgaaggacc
     1261 gcgcacctgg tgcatgaccc gcaagcccgg tgcctgacgc ccgccccacg acccgcagcg
     1321 cccgaccgaa aggagcgcac gaccccatgc atcgattagt ccaatttgtt aaagacagga
     1381 tatcagtggt ccaggctcta gttttgactc aacaatatca ccagctgaag cctatagagt
     1441 acgagccata gataaaataa aagattttat ttagtctcca gaaaaagggg ggaatgaaag
     1501 accccacctg taggtttggc aagctagctt aagtaacgcc attttgcaag gcatggaaaa
     1561 atacataact gagaatagag aagttcagat caaggtcagg aacagatgga acagggtcga
     1621 ccctagagaa ccatcagatg tttccagggt gccccaagga cctgaaatga ccctgtgcct
     1681 tatttgaact aaccaatcag ttcgcttctc gcttctgttc gcgcgcttct gctccccgag
     1741 ctcaataaaa gagcccacaa cccctcactc ggggcgccag tcctccgatt gactgagtcg
     1801 cccgggtacc cgtgtatcca ataaaccctc ttgcagttgc atccgacttg tggtctcgct
     1861 gttccttggg agggtctcct ctgagtgatt gactacccgt cagcgggggt ctttcatttg
     1921 ggggctcgtc cgggatcggg agacccctgc ccagggacca ccgacccacc accgggaggt
     1981 aagctggctg cctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc
     2041 cggagacggt cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg
     2101 cgtcagcggg tgttggcggg tgtcggggcg cagccatgac ccagtcacgt agcgatagcg
     2161 gagtgtagat ccggctgtgg aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc
     2221 cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccagg tgtggaaagt
     2281 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
     2341 tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc
     2401 cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc tcggcctctg
     2461 agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaaaagctta
     2521 ctggcttaac tatgcggcat cagagcagat tgtactgaga gtgcaccata tgcggtgtga
     2581 aataccgcac agatgcgtaa ggagaaaata ccgcatcagg cgctcttccg cttcctcgct
     2641 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
     2701 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     2761 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     2821 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     2881 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     2941 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     3001 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     3061 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     3121 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     3181 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     3241 tagaaggaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     3301 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     3361 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     3421 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     3481 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     3541 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     3601 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     3661 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
     3721 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     3781 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     3841 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct gcaggcatcg tggtgtcacg
     3901 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     3961 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     4021 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     4081 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     4141 atagtgtatg cggcgaccga gttgctcttg cccggcgtca acacgggata ataccgcgcc
     4201 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     4261 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     4321 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     4381 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     4441 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     4501 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt
     4561 ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca cgaggccctt
     4621 tcgtcttcaa gaattagctt ggccattgca tacgttgtat ccatatcata atatgtacat
     4681 ttatattggc tcatgtccaa cattaccgcc atgttgacat tgattattga ctagttatta
     4741 atagtaatca attacggggt cattagttca tagcccatat atggagttcc gcgttacata
     4801 acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat
     4861 aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga
     4921 gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc
     4981 ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt
     5041 atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat
     5101 gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag
     5161 tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc
     5221 aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga
     5281 ggtctatata agcagagctc aataaaagag cccacaaccc ctcactcggc gcgccagtct
     5341 tccgatagac tgcgtcgccc gggtacccgt attcccaata aagcctcttg ctgtttgcat
     5401 ccgaatcgtg gtctcgctgt tccttgggag ggtctcctct gagtgattga ctacccacga
     5461 cgggggtctt tcatttgggg gctcgtccgg gatttggaga cccctgccca gggaccaccg
     5521 acccaccacc gggaggtaag ctggccagca acttatctgt gtctgtccga ttgtctagtg
     5581 tctatgtttg atgttatgcg cctgcgtctg tactagttag ctaactagct ctgtatctgg
     5641 cggacccgtg gtggaactga cgagttctga acacccggcc gcaaccctgg gagacgtccc
     5701 agggactttg ggggccgttt ttgtggcccg acctgaggaa gggagtcgat gtggaatccg
     5761 accccgtcag gatatgtggt tctggtagga gacgagaacc taaaacagtt cccgcctccg
     5821 tctgaatttt tgctttcggt ttggaaccga agccgcgcgt cttgtctgct gcagcgctgc
     5881 agcatcgttc tgtgttgtct ctgtctgact gtgtttctgt atttgtctga aaattagggc
     5941 cagactgtta ccactccctt aagtttgacc ttaggtcact ggaaagatgt cgagcggatc
     6001 gctcacaacc agtcggtaga tgtcaagaag agacgttggg ttaccttctg ctctgcagaa
     6061 tggccaacct ttaacgtcgg atggccgcga gacggcacct ttaaccgaga cctcatcacc
     6121 caggttaaga tcaaggtctt ttcacctggc ccgcatggac acccagacca ggtcccctac
     6181 atcgtgacct gggaagcctt ggcttttgac ccccctccct gggtcaagcc ctttgtacac
     6241 cctaagcctc cgcctcctct tcctccatcc gccccgtctc tcccccttga acctcctcgt
     6301 tcgaccccgc ctcgatcctc cctttatcca gccctcactc cttctctagg cgccggaatt
     6361 gaagatctgt c
//
