LOCUS       sequence-412264-        8031 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Entry cloning vector to insert an sgRNA of interest (using Esp3i 
            digestion) into a vector that already contains sgRNAs against mouse 
            Rb1, Trp53, and Rbl2 and CMV Cre recombinase..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8031)
  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 8031)
  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-sgX-U6-sgRb1-U6-sgTrp53-U6-sgRbl2
FEATURES             Location/Qualifiers
     source          1..8031
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(97..124)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(216..302)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     351..373
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     365..382
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     366..382
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    398..497
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(555..581)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             complement(1608..1628)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     promoter        complement(1648..1851)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(1677..1697)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        1852..2155
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     protein_bind    2206..2226
                     /gene="mutant version of attB"
                     /label=attB5
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     promoter        2305..2553
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        4435..4510
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        4530..4778
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        4798..4873
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(4933..4953)
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     misc_RNA        5215..5290
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    5338..5358
                     /gene="mutant version of attB"
                     /label=attB3
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     misc_RNA        5618..5693
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(5739..5838)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(5855..5874)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5856..5874)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5879..5895)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5879..5895)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             6326..7117
                     /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      7210..7798
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7699..7718
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7952..7969
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatac
       61 gcgtaccgct agccaggaag agtttgtaga aacgcaaaaa ggccatccgt caggatggcc
      121 ttctgcttag tttgatgcct ggcagtttat ggcgggcgtc ctgcccgcca ccctccgggc
      181 cgttgcttca caacgttcaa atccgctccc ggcggatttg tcctactcag gagagcgttc
      241 accgacaaac aacagataaa acgaaaggcc cagtcttccg actgagcctt tcgttttatt
      301 tgatgcctgg cagttcccta ctctcgcgtt aacgctagca tggatgtttt cccagtcacg
      361 acgttgtaaa acgacggcca gtcttaagct cgggccccaa ataatgattt tattttgact
      421 gatagtgacc tgttcgttgc aacaaattga tgagcaatgc ttttttataa tgccaacttt
      481 gtacaaaaaa gcaggctaca caaaaaacca acacacagat ctaatgaaaa taaagatctt
      541 ttattagcgc tttaagcgta atctggaaca tcgtatgggt agtcaccatc ttcgagcagt
      601 ctcaccatgg ccccggtttc agagtcaaga ttgcggatat agttcataac gatgttcaca
      661 tttgtccaac ctccagcttg cataatctct ggtatggaga ctcctgcccg cgccatgtct
      721 cgtgcggccc ccaccctagc agaatgacct gaccaggcga gatacctctg gccactgtca
      781 tccttggctc cgtatatgag tcggtgggtt gcctcaaaga tcccttcaag ggcccgtgta
      841 ctcaattggc tggtggcaga gggcgcggcg actccgtttt ttctcactcg acagaacaag
      901 taattattag ggtcgtcagc cacgccggac actgagatcc atctttccac cagcttcgtt
      961 acgcccaggg acaaggcctt ttcaacgcca gcggtgctga ccaatgtctt tgtgcgtcca
     1021 atatgaataa gcatccggcc gccgtccgtt cgactgatgt ctttcactct gatccgggcg
     1081 atctcagcta tgcgcagaag tgtgttatat gcaattccca gaaatgcgag gttcctgatg
     1141 tcctggcacc gatcgctgtt ctccatcagg gatctgacct ggtcgaaatc agtccgctca
     1201 aaagcgagag cctgcttagc cctctcaccc gcatctacgt tttcctttct aattctcctc
     1261 ataacaaggg agactgcgtt tgaatcgcta ggccggggga gcccgctgcg tcgatgcagc
     1321 atattcagtt ggcccaagtg ctgctggata gtcttgacag ccagccctct tgcctggaga
     1381 tacagcaggt aatcgcgcac gtcctccggc tcggcgggga accatttgcg attgttcagc
     1441 ttacaccagg ctgcccagga tcggcacacg ctaagcagca ttttccaggt gtgttcagaa
     1501 aacgcctgtc tatccctaaa catatccatg agatttttcc tgacctcatc gctagtggca
     1561 tctactggca gcgcagggag gttctggtga acagtcagga gattggacac cttcctcttc
     1621 ttcttgggca ttgacggttc ataaaccagc tctgcttata tagacctccc accgtacacg
     1681 cctaccgccc atttgcgtca atggggcgga gttgttacga cattttggaa agtcccgttg
     1741 attttggtgc caaaacaaac tcccattgac gtcaatgggg tggagacttg gaaatccccg
     1801 tgagtcaaac cgctatccac gcccattgat gtactgccaa aaccgcatca ccatggtaat
     1861 agcgatgact aatacgtaga tgtactgcca agtaggaaag tcccataagg tcatgtactg
     1921 ggcataatgc caggcgggcc atttaccgtc attgacgtca atagggggcg tacttggcat
     1981 atgatacact tgatgtactg ccaagtgggc agtttaccgt aaatactcca cccattgacg
     2041 tcaatggaaa gtccctattg gcgttactat gggaacatac gtcattattg acgtcaatgg
     2101 gcgggggtcg ttgggcggtc agccaggcgg gccatttacc gtaagttatg taacgcggaa
     2161 ctccatatat gggctatgaa ctaatgaccc cgtaattgat tactacaact ttgtatacaa
     2221 aagttgtggt tattttggat ccaccggtat cgatgaattc aagcttggta ccgtcgacga
     2281 tccagtttgg ttcgttaagg taccgagggc ctatttccca tgattccttc atatttgcat
     2341 atacgataca aggctgttag agagataatt agaattaatt tgactgtaaa cacaaagata
     2401 ttagtacaaa atacgtgacg tagaaagtaa taatttcttg ggtagtttgc agttttaaaa
     2461 ttatgtttta aaatggacta tcatatgctt accgtaactt gaaagtattt cgatttcttg
     2521 gctttatata tcttgtggaa aggacgaaac accggagacg gttgtaaatg agcacacaaa
     2581 atacacatgc taaaatatta tattctatga cctttataaa atcaaccaaa atcttctttt
     2641 taataacttt agtatcaata attagaattt ttatgttcct ttttgcaaac ttttaataaa
     2701 aatgagcaaa ataaaaaaac gctagtttta gtaactcgcg ttgttttctt cacctttaat
     2761 aatagctact ccaccacttg ttcctaagcg gtcagctcct gcttcaatca ttttttgagc
     2821 atcttcaaat gttctaactc caccagctgc tttaactaaa gcattgtctt taacaactga
     2881 cttcattagt ttaacatctt caaatgttgc acctgatttt gaaaatcctg ttgatgtttt
     2941 aacaaattct aatccagctt caacagctat ttcacaagct ttcatgattt cttcttttgt
     3001 taataaacaa ttttccataa tacatttaac aacatgtgat ccagctgctt tttttacagc
     3061 tttcatgtct tctaaaacta attcataatt tttgtctttt aatgcaccaa tatttaatac
     3121 catatcaatt tctgttgcac catctttaat tgcttcagaa acttcgaatg cttttgtagc
     3181 tgttgtgcat gcacctagag gaaaacctac aacatttgtt attcctacat ttgtgccttt
     3241 taataattct ttacaatagc ttgttcaata tgaattaaca caaactgttg caaaatcaaa
     3301 ttcaattgct tcatcacata attgtttaat ttcagctttc gtagcatctt gttttaataa
     3361 tgtgtgatct atatatttgt ttagtttcat tttttctcct atatattcat ttttaatttt
     3421 aattctttaa taatttcgtc tactttaact ttagcgtttt gaacagattc accaacacct
     3481 ataaaataaa tttttagttt aggttcagtt ccacttgggc gaacagcaaa tcatgactta
     3541 tcttctaaat aaaattttag taagtcttgt cctggcatat tatacattcc atcgatgtag
     3601 tcttcaacat taacaacttt aagtccagca atttgagtta agggtgttgc tctcaatgat
     3661 ttcattaatg gttcaatttt taatttcttt tcttctggtt taaaattcaa gtttaaagtg
     3721 aaagtgtaat atgcacccat ttctttaaat aaatcttcta aatagtctac taatgtttta
     3781 ttttgttttt tataaaatca agcagcctct gctattaata tagaagcttg tattccatct
     3841 ttatctctag ctgagtcatc aattacatat ccataacttt cttcataagc aaaaacaaaa
     3901 tttaatccgt tatcttcttc tttagcaatt tctctaccca ttcatttaaa tccagttaaa
     3961 gtttttacaa tattaactcc atatttttca tgagcgattc tatcacccaa atcacttgtt
     4021 acaaaacttg aatatagagc cggatttttt ggaatgctat ttaagcgttt tagatttgat
     4081 aattttcaat caattaaaat tggtcctgtt tgatttccat ctaatcttac aaaatgacca
     4141 tcatgtttta ttgccattcc aaatctgtca gcatctgggt cattcataat aataatatct
     4201 gcatcatgtt taataccata ttcaagcggt atttttcatg caggatcaaa ttctggattt
     4261 ggatttacaa catttttaaa tgtttcatct tcaaatgcat gctcttcaac ctcaataacg
     4321 ttatatcctg attcacgtaa tatttttggg gtaaatttag ttcctgttcc attaactgcg
     4381 ctaaaaataa tttttaaatc ttttttagct tcttgctctt ttttgtacgt ctctgtttta
     4441 gagctagaaa tagcaagtta aaataaggct agtccgttat caacttgaaa aagtggcacc
     4501 gagtcggtgc ttttttgaat tcgctagctg agggcctatt tcccatgatt ccttcatatt
     4561 tgcatatacg atacaaggct gttagagaga taattagaat taatttgact gtaaacacaa
     4621 agatattagt acaaaatacg tgacgtagaa agtaataatt tcttgggtag tttgcagttt
     4681 taaaattatg ttttaaaatg gactatcata tgcttaccgt aacttgaaag tatttcgatt
     4741 tcttggcttt atatatcttg tggaaaggac gaaacaccgc aactagaaaa tgatacggtt
     4801 ttagagctag aaatagcaag ttaaaataag gctagtccgt tatcaacttg aaaaagtggc
     4861 accgagtcgg tgcttttttg aattcgagct cccgcggtct agacccggga ctagtagatc
     4921 tctcgagaca cccaactttt ctatacaaag ttgtttccca tgattccttc atatttgcat
     4981 atacgataca aggctgttag agagataatt agaattaatt tgactgtaaa cacaaagata
     5041 ttagtacaaa atacgtgacg tagaaagtaa taatttcttg ggtagtttgc agttttaaaa
     5101 ttatgtttta aaatggacta tcatatgctt accgtaactt gaaagtattt cgatttcttg
     5161 gctttatata tcttgtggaa aggacgaaac accggtgtaa tagctcctgc atgggtttta
     5221 gagctagaaa tagcaagtta aaataaggct agtccgttat caacttgaaa aagtggcacc
     5281 gagtcggtgc ttttttgaat tctagatctt gagacaaatg gcagtattca tccacaacaa
     5341 ctttgtataa taaagttgtt tcccatgatt ccttcatatt tgcatatacg atacaaggct
     5401 gttagagaga taattagaat taatttgact gtaaacacaa agatattagt acaaaatacg
     5461 tgacgtagaa agtaataatt tcttgggtag tttgcagttt taaaattatg ttttaaaatg
     5521 gactatcata tgcttaccgt aacttgaaag tatttcgatt tcttggcttt atatatcttg
     5581 tggaaaggac gaaacaccga ggaggatggc gacgccggtt ttagagctag aaatagcaag
     5641 ttaaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttg
     5701 aattctagat cttgagacaa atggcagtat tcatccacac ccagctttct tgtacaaagt
     5761 tggcattata agaaagcatt gcttatcaat ttgttgcaac gaacaggtca ctatcagtca
     5821 aaataaaatc attatttgcc atccagctga tatcccctat agtgagtcgt attacatggt
     5881 catagctgtt tcctggcagc tctggcccgt gtctcaaaat ctctgatgtt acattgcaca
     5941 agataaaaat atatcatcat gcctcctcta gaccagccag gacagaaatg cctcgacttc
     6001 gctgctaccc aaggttgccg ggtgacgcac accgtggaaa cggatgaagg cacgaaccca
     6061 gtggacataa gcctgttcgg ttcgtaagct gtaatgcaag tagcgtatgc gctcacgcaa
     6121 ctggtccaga accttgaccg aacgcagcgg tggtaacggc gcagtggcgg ttttcatggc
     6181 ttgttatgac tgtttttttg gggtacagtc tatgcctcgg gcatccaagc agcaagcgcg
     6241 ttacgccgtg ggtcgatgtt tgatgttatg gagcagcaac gatgttacgc agcagggcag
     6301 tcgccctaaa acaaagttaa acattatgag ggaagcggtg atcgccgaag tatcgactca
     6361 actatcagag gtagttggcg tcatcgagcg ccatctcgaa ccgacgttgc tggccgtaca
     6421 tttgtacggc tccgcagtgg atggcggcct gaagccacac agtgatattg atttgctggt
     6481 tacggtgacc gtaaggcttg atgaaacaac gcggcgagct ttgatcaacg accttttgga
     6541 aacttcggct tcccctggag agagcgagat tctccgcgct gtagaagtca ccattgttgt
     6601 gcacgacgac atcattccgt ggcgttatcc agctaagcgc gaactgcaat ttggagaatg
     6661 gcagcgcaat gacattcttg caggtatctt cgagccagcc acgatcgaca ttgatctggc
     6721 tatcttgctg acaaaagcaa gagaacatag cgttgccttg gtaggtccag cggcggagga
     6781 actctttgat ccggttcctg aacaggatct atttgaggcg ctaaatgaaa ccttaacgct
     6841 atggaactcg ccgcccgact gggctggcga tgagcgaaat gtagtgctta cgttgtcccg
     6901 catttggtac agcgcagtaa ccggcaaaat cgcgccgaag gatgtcgctg ccgactgggc
     6961 aatggagcgc ctgccggccc agtatcagcc cgtcatactt gaagctagac aggcttatct
     7021 tggacaagaa gaagatcgct tggcctcgcg cgcagatcag ttggaagaat ttgtccacta
     7081 cgtgaaaggc gagatcacca aggtagtcgg caaataaccc tcgagccacc catgaccaaa
     7141 atcccttaac gtgagttacg cgtcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     7201 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     7261 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     7321 aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg
     7381 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     7441 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     7501 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     7561 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     7621 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     7681 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     7741 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     7801 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
     7861 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
     7921 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
     7981 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat g
//
