LOCUS       sequence-417708-        6169 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Entry vector that encodes sgRNAs against mouse Rb1, Trp53, Rbl2, and
            a non-targeting sgRNA (C0111) and CMV Cre recombinase..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6169)
  AUTHORS   Duplaquet L, Li Y, Booker MA, Xie Y, Olsen SN, Patel RA, Hong D, 
            Hatton C, Denize T, Walton E, Laimon YN, Li R, Jiang Y, Bronson RT, 
            Southard J, Li S, Signoretti S, Qiu X, Cejas P, Armstrong SA, Long 
            HW, Tolstorukov MY, Haffner MC, Oser MG
  TITLE     KDM6A epigenetically regulates subtype plasticity in small cell lung
            cancer.
  JOURNAL   Nat Cell Biol. 2023 Sep;25(9):1346-1358. doi: 
            10.1038/s41556-023-01210-z. Epub 2023 Aug 17.
  PUBMED    37591951
REFERENCE   2  (bases 1 to 6169)
  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: 
            "10.1038/s41556-023-01210-z"; journalName: "Nat Cell Biol"; date: 
            "2023-09"; volume: "25"; issue: "9"; pages: "1346-1358"
COMMENT     Sequence Label: 
            pENTR223-CMV-Cre-U6-sgC0111-U6-sgRb1-U6-sgTrp53-U6-sgRbl2
FEATURES             Location/Qualifiers
     source          1..6169
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(149..176)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(268..354)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     403..425
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     417..434
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     418..434
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    450..549
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(607..633)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             complement(1660..1680)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     promoter        complement(1700..1903)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(1729..1749)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        1904..2207
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     protein_bind    2258..2278
                     /gene="mutant version of attB"
                     /label=attB5
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     promoter        2357..2605
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        2625..2700
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        2720..2968
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        2988..3063
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(3123..3143)
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     misc_RNA        3405..3480
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    3528..3548
                     /gene="mutant version of attB"
                     /label=attB3
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     misc_RNA        3808..3883
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(3929..4028)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(4045..4064)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4046..4064)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4069..4085)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4069..4085)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             4516..5307
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     rep_origin      5400..5988
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5889..5908
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6142..6159
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
       61 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat acgcgtaccg
      121 ctagccagga agagtttgta gaaacgcaaa aaggccatcc gtcaggatgg ccttctgctt
      181 agtttgatgc ctggcagttt atggcgggcg tcctgcccgc caccctccgg gccgttgctt
      241 cacaacgttc aaatccgctc ccggcggatt tgtcctactc aggagagcgt tcaccgacaa
      301 acaacagata aaacgaaagg cccagtcttc cgactgagcc tttcgtttta tttgatgcct
      361 ggcagttccc tactctcgcg ttaacgctag catggatgtt ttcccagtca cgacgttgta
      421 aaacgacggc cagtcttaag ctcgggcccc aaataatgat tttattttga ctgatagtga
      481 cctgttcgtt gcaacaaatt gatgagcaat gcttttttat aatgccaact ttgtacaaaa
      541 aagcaggcta cacaaaaaac caacacacag atctaatgaa aataaagatc ttttattagc
      601 gctttaagcg taatctggaa catcgtatgg gtagtcacca tcttcgagca gtctcaccat
      661 ggccccggtt tcagagtcaa gattgcggat atagttcata acgatgttca catttgtcca
      721 acctccagct tgcataatct ctggtatgga gactcctgcc cgcgccatgt ctcgtgcggc
      781 ccccacccta gcagaatgac ctgaccaggc gagatacctc tggccactgt catccttggc
      841 tccgtatatg agtcggtggg ttgcctcaaa gatcccttca agggcccgtg tactcaattg
      901 gctggtggca gagggcgcgg cgactccgtt ttttctcact cgacagaaca agtaattatt
      961 agggtcgtca gccacgccgg acactgagat ccatctttcc accagcttcg ttacgcccag
     1021 ggacaaggcc ttttcaacgc cagcggtgct gaccaatgtc tttgtgcgtc caatatgaat
     1081 aagcatccgg ccgccgtccg ttcgactgat gtctttcact ctgatccggg cgatctcagc
     1141 tatgcgcaga agtgtgttat atgcaattcc cagaaatgcg aggttcctga tgtcctggca
     1201 ccgatcgctg ttctccatca gggatctgac ctggtcgaaa tcagtccgct caaaagcgag
     1261 agcctgctta gccctctcac ccgcatctac gttttccttt ctaattctcc tcataacaag
     1321 ggagactgcg tttgaatcgc taggccgggg gagcccgctg cgtcgatgca gcatattcag
     1381 ttggcccaag tgctgctgga tagtcttgac agccagccct cttgcctgga gatacagcag
     1441 gtaatcgcgc acgtcctccg gctcggcggg gaaccatttg cgattgttca gcttacacca
     1501 ggctgcccag gatcggcaca cgctaagcag cattttccag gtgtgttcag aaaacgcctg
     1561 tctatcccta aacatatcca tgagattttt cctgacctca tcgctagtgg catctactgg
     1621 cagcgcaggg aggttctggt gaacagtcag gagattggac accttcctct tcttcttggg
     1681 cattgacggt tcataaacca gctctgctta tatagacctc ccaccgtaca cgcctaccgc
     1741 ccatttgcgt caatggggcg gagttgttac gacattttgg aaagtcccgt tgattttggt
     1801 gccaaaacaa actcccattg acgtcaatgg ggtggagact tggaaatccc cgtgagtcaa
     1861 accgctatcc acgcccattg atgtactgcc aaaaccgcat caccatggta atagcgatga
     1921 ctaatacgta gatgtactgc caagtaggaa agtcccataa ggtcatgtac tgggcataat
     1981 gccaggcggg ccatttaccg tcattgacgt caataggggg cgtacttggc atatgataca
     2041 cttgatgtac tgccaagtgg gcagtttacc gtaaatactc cacccattga cgtcaatgga
     2101 aagtccctat tggcgttact atgggaacat acgtcattat tgacgtcaat gggcgggggt
     2161 cgttgggcgg tcagccaggc gggccattta ccgtaagtta tgtaacgcgg aactccatat
     2221 atgggctatg aactaatgac cccgtaattg attactacaa ctttgtatac aaaagttgtg
     2281 gttattttgg atccaccggt atcgatgaat tcaagcttgg taccgtcgac gatccagttt
     2341 ggttcgttaa ggtaccgagg gcctatttcc catgattcct tcatatttgc atatacgata
     2401 caaggctgtt agagagataa ttagaattaa tttgactgta aacacaaaga tattagtaca
     2461 aaatacgtga cgtagaaagt aataatttct tgggtagttt gcagttttaa aattatgttt
     2521 taaaatggac tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct tggctttata
     2581 tatcttgtgg aaaggacgaa acaccgggag gctaagcgtc gcaagtttta gagctagaaa
     2641 tagcaagtta aaataaggct agtccgttat caacttgaaa aagtggcacc gagtcggtgc
     2701 ttttttgaat tcgctagctg agggcctatt tcccatgatt ccttcatatt tgcatatacg
     2761 atacaaggct gttagagaga taattagaat taatttgact gtaaacacaa agatattagt
     2821 acaaaatacg tgacgtagaa agtaataatt tcttgggtag tttgcagttt taaaattatg
     2881 ttttaaaatg gactatcata tgcttaccgt aacttgaaag tatttcgatt tcttggcttt
     2941 atatatcttg tggaaaggac gaaacaccgc aactagaaaa tgatacggtt ttagagctag
     3001 aaatagcaag ttaaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg
     3061 tgcttttttg aattcgagct cccgcggtct agacccggga ctagtagatc tctcgagaca
     3121 cccaactttt ctatacaaag ttgtttccca tgattccttc atatttgcat atacgataca
     3181 aggctgttag agagataatt agaattaatt tgactgtaaa cacaaagata ttagtacaaa
     3241 atacgtgacg tagaaagtaa taatttcttg ggtagtttgc agttttaaaa ttatgtttta
     3301 aaatggacta tcatatgctt accgtaactt gaaagtattt cgatttcttg gctttatata
     3361 tcttgtggaa aggacgaaac accggtgtaa tagctcctgc atgggtttta gagctagaaa
     3421 tagcaagtta aaataaggct agtccgttat caacttgaaa aagtggcacc gagtcggtgc
     3481 ttttttgaat tctagatctt gagacaaatg gcagtattca tccacaacaa ctttgtataa
     3541 taaagttgtt tcccatgatt ccttcatatt tgcatatacg atacaaggct gttagagaga
     3601 taattagaat taatttgact gtaaacacaa agatattagt acaaaatacg tgacgtagaa
     3661 agtaataatt tcttgggtag tttgcagttt taaaattatg ttttaaaatg gactatcata
     3721 tgcttaccgt aacttgaaag tatttcgatt tcttggcttt atatatcttg tggaaaggac
     3781 gaaacaccga ggaggatggc gacgccggtt ttagagctag aaatagcaag ttaaaataag
     3841 gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttg aattctagat
     3901 cttgagacaa atggcagtat tcatccacac ccagctttct tgtacaaagt tggcattata
     3961 agaaagcatt gcttatcaat ttgttgcaac gaacaggtca ctatcagtca aaataaaatc
     4021 attatttgcc atccagctga tatcccctat agtgagtcgt attacatggt catagctgtt
     4081 tcctggcagc tctggcccgt gtctcaaaat ctctgatgtt acattgcaca agataaaaat
     4141 atatcatcat gcctcctcta gaccagccag gacagaaatg cctcgacttc gctgctaccc
     4201 aaggttgccg ggtgacgcac accgtggaaa cggatgaagg cacgaaccca gtggacataa
     4261 gcctgttcgg ttcgtaagct gtaatgcaag tagcgtatgc gctcacgcaa ctggtccaga
     4321 accttgaccg aacgcagcgg tggtaacggc gcagtggcgg ttttcatggc ttgttatgac
     4381 tgtttttttg gggtacagtc tatgcctcgg gcatccaagc agcaagcgcg ttacgccgtg
     4441 ggtcgatgtt tgatgttatg gagcagcaac gatgttacgc agcagggcag tcgccctaaa
     4501 acaaagttaa acattatgag ggaagcggtg atcgccgaag tatcgactca actatcagag
     4561 gtagttggcg tcatcgagcg ccatctcgaa ccgacgttgc tggccgtaca tttgtacggc
     4621 tccgcagtgg atggcggcct gaagccacac agtgatattg atttgctggt tacggtgacc
     4681 gtaaggcttg atgaaacaac gcggcgagct ttgatcaacg accttttgga aacttcggct
     4741 tcccctggag agagcgagat tctccgcgct gtagaagtca ccattgttgt gcacgacgac
     4801 atcattccgt ggcgttatcc agctaagcgc gaactgcaat ttggagaatg gcagcgcaat
     4861 gacattcttg caggtatctt cgagccagcc acgatcgaca ttgatctggc tatcttgctg
     4921 acaaaagcaa gagaacatag cgttgccttg gtaggtccag cggcggagga actctttgat
     4981 ccggttcctg aacaggatct atttgaggcg ctaaatgaaa ccttaacgct atggaactcg
     5041 ccgcccgact gggctggcga tgagcgaaat gtagtgctta cgttgtcccg catttggtac
     5101 agcgcagtaa ccggcaaaat cgcgccgaag gatgtcgctg ccgactgggc aatggagcgc
     5161 ctgccggccc agtatcagcc cgtcatactt gaagctagac aggcttatct tggacaagaa
     5221 gaagatcgct tggcctcgcg cgcagatcag ttggaagaat ttgtccacta cgtgaaaggc
     5281 gagatcacca aggtagtcgg caaataaccc tcgagccacc catgaccaaa atcccttaac
     5341 gtgagttacg cgtcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     5401 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     5461 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     5521 agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc
     5581 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     5641 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     5701 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     5761 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     5821 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     5881 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     5941 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     6001 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     6061 ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc
     6121 cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaag
//
