LOCUS       sequence-417712-        6170 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Entry vector that endcodes sgRNAs against mouse Kdm6a, Rb1, Trp53, 
            and Rbl2 and CMV Cre recombinase..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6170)
  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 6170)
  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-sgKdm6a#4-U6-sgRb1-U6-sgTrp53-U6-sgRbl2
FEATURES             Location/Qualifiers
     source          1..6170
                     /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        2626..2701
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        2721..2969
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        2989..3064
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(3124..3144)
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     misc_RNA        3406..3481
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    3529..3549
                     /gene="mutant version of attB"
                     /label=attB3
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     misc_RNA        3809..3884
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(3930..4029)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(4046..4065)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4047..4065)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4070..4086)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4070..4086)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             4517..5308
                     /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      5401..5989
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5890..5909
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6143..6160
                     /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 acaccgcctt ggctcgacaa aagctgtttt agagctagaa
     2641 atagcaagtt aaaataaggc tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg
     2701 cttttttgaa ttcgctagct gagggcctat ttcccatgat tccttcatat ttgcatatac
     2761 gatacaaggc tgttagagag ataattagaa ttaatttgac tgtaaacaca aagatattag
     2821 tacaaaatac gtgacgtaga aagtaataat ttcttgggta gtttgcagtt ttaaaattat
     2881 gttttaaaat ggactatcat atgcttaccg taacttgaaa gtatttcgat ttcttggctt
     2941 tatatatctt gtggaaagga cgaaacaccg caactagaaa atgatacggt tttagagcta
     3001 gaaatagcaa gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg
     3061 gtgctttttt gaattcgagc tcccgcggtc tagacccggg actagtagat ctctcgagac
     3121 acccaacttt tctatacaaa gttgtttccc atgattcctt catatttgca tatacgatac
     3181 aaggctgtta gagagataat tagaattaat ttgactgtaa acacaaagat attagtacaa
     3241 aatacgtgac gtagaaagta ataatttctt gggtagtttg cagttttaaa attatgtttt
     3301 aaaatggact atcatatgct taccgtaact tgaaagtatt tcgatttctt ggctttatat
     3361 atcttgtgga aaggacgaaa caccggtgta atagctcctg catgggtttt agagctagaa
     3421 atagcaagtt aaaataaggc tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg
     3481 cttttttgaa ttctagatct tgagacaaat ggcagtattc atccacaaca actttgtata
     3541 ataaagttgt ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
     3601 ataattagaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
     3661 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
     3721 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
     3781 cgaaacaccg aggaggatgg cgacgccggt tttagagcta gaaatagcaa gttaaaataa
     3841 ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt gaattctaga
     3901 tcttgagaca aatggcagta ttcatccaca cccagctttc ttgtacaaag ttggcattat
     3961 aagaaagcat tgcttatcaa tttgttgcaa cgaacaggtc actatcagtc aaaataaaat
     4021 cattatttgc catccagctg atatccccta tagtgagtcg tattacatgg tcatagctgt
     4081 ttcctggcag ctctggcccg tgtctcaaaa tctctgatgt tacattgcac aagataaaaa
     4141 tatatcatca tgcctcctct agaccagcca ggacagaaat gcctcgactt cgctgctacc
     4201 caaggttgcc gggtgacgca caccgtggaa acggatgaag gcacgaaccc agtggacata
     4261 agcctgttcg gttcgtaagc tgtaatgcaa gtagcgtatg cgctcacgca actggtccag
     4321 aaccttgacc gaacgcagcg gtggtaacgg cgcagtggcg gttttcatgg cttgttatga
     4381 ctgttttttt ggggtacagt ctatgcctcg ggcatccaag cagcaagcgc gttacgccgt
     4441 gggtcgatgt ttgatgttat ggagcagcaa cgatgttacg cagcagggca gtcgccctaa
     4501 aacaaagtta aacattatga gggaagcggt gatcgccgaa gtatcgactc aactatcaga
     4561 ggtagttggc gtcatcgagc gccatctcga accgacgttg ctggccgtac atttgtacgg
     4621 ctccgcagtg gatggcggcc tgaagccaca cagtgatatt gatttgctgg ttacggtgac
     4681 cgtaaggctt gatgaaacaa cgcggcgagc tttgatcaac gaccttttgg aaacttcggc
     4741 ttcccctgga gagagcgaga ttctccgcgc tgtagaagtc accattgttg tgcacgacga
     4801 catcattccg tggcgttatc cagctaagcg cgaactgcaa tttggagaat ggcagcgcaa
     4861 tgacattctt gcaggtatct tcgagccagc cacgatcgac attgatctgg ctatcttgct
     4921 gacaaaagca agagaacata gcgttgcctt ggtaggtcca gcggcggagg aactctttga
     4981 tccggttcct gaacaggatc tatttgaggc gctaaatgaa accttaacgc tatggaactc
     5041 gccgcccgac tgggctggcg atgagcgaaa tgtagtgctt acgttgtccc gcatttggta
     5101 cagcgcagta accggcaaaa tcgcgccgaa ggatgtcgct gccgactggg caatggagcg
     5161 cctgccggcc cagtatcagc ccgtcatact tgaagctaga caggcttatc ttggacaaga
     5221 agaagatcgc ttggcctcgc gcgcagatca gttggaagaa tttgtccact acgtgaaagg
     5281 cgagatcacc aaggtagtcg gcaaataacc ctcgagccac ccatgaccaa aatcccttaa
     5341 cgtgagttac gcgtcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     5401 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     5461 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     5521 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
     5581 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     5641 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     5701 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     5761 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     5821 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     5881 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     5941 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     6001 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
     6061 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
     6121 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag
//
