LOCUS       sequence-412278-        6171 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Entry vector that encodes sgRNAs against mouse Notch2, Rb1, Trp53, 
            Rbl2, and CMV Cre recombinase..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6171)
  AUTHORS   Hong D, Knelson EH, Li Y, Durmaz YT, Gao W, Walton E, Vajdi A, Thai 
            T, Sticco-Ivins M, Sabet AH, Jones KL, Schinzel AC, Bronson RT, 
            Nguyen QD, Tolstorukov MY, Vivero M, Signoretti S, Barbie DA, Oser 
            MG
  TITLE     Plasticity in the Absence of NOTCH Uncovers a RUNX2-Dependent 
            Pathway in Small Cell Lung Cancer.
  JOURNAL   Cancer Res. 2022 Jan 15;82(2):248-263. doi: 
            10.1158/0008-5472.CAN-21-1991. Epub 2021 Nov 22.
  PUBMED    34810201
REFERENCE   2  (bases 1 to 6171)
  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: 
            "10.1158/0008-5472.CAN-21-1991"; journalName: "Cancer Res"; date: 
            "2022-01-15- 15"; volume: "82"; issue: "2"; pages: "248-263"
COMMENT     Sequence Label: 
            pENTR223-CMV-Cre-U6-sgNotch2#1-U6-sgRb1-U6-sgTrp53-U6-sgRbl2
FEATURES             Location/Qualifiers
     source          1..6171
                     /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        2575..2650
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        2670..2918
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        2938..3013
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(3073..3093)
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     misc_RNA        3355..3430
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    3478..3498
                     /gene="mutant version of attB"
                     /label=attB3
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     misc_RNA        3758..3833
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(3879..3978)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(3995..4014)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3996..4014)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4019..4035)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4019..4035)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             4466..5257
                     /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      5350..5938
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5839..5858
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6092..6109
                     /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 accgctctgc gtcccgccgc gctggtttta
     2581 gagctagaaa tagcaagtta aaataaggct agtccgttat caacttgaaa aagtggcacc
     2641 gagtcggtgc ttttttgaat tcgctagctg agggcctatt tcccatgatt ccttcatatt
     2701 tgcatatacg atacaaggct gttagagaga taattagaat taatttgact gtaaacacaa
     2761 agatattagt acaaaatacg tgacgtagaa agtaataatt tcttgggtag tttgcagttt
     2821 taaaattatg ttttaaaatg gactatcata tgcttaccgt aacttgaaag tatttcgatt
     2881 tcttggcttt atatatcttg tggaaaggac gaaacaccgc aactagaaaa tgatacggtt
     2941 ttagagctag aaatagcaag ttaaaataag gctagtccgt tatcaacttg aaaaagtggc
     3001 accgagtcgg tgcttttttg aattcgagct cccgcggtct agacccggga ctagtagatc
     3061 tctcgagaca cccaactttt ctatacaaag ttgtttccca tgattccttc atatttgcat
     3121 atacgataca aggctgttag agagataatt agaattaatt tgactgtaaa cacaaagata
     3181 ttagtacaaa atacgtgacg tagaaagtaa taatttcttg ggtagtttgc agttttaaaa
     3241 ttatgtttta aaatggacta tcatatgctt accgtaactt gaaagtattt cgatttcttg
     3301 gctttatata tcttgtggaa aggacgaaac accggtgtaa tagctcctgc atgggtttta
     3361 gagctagaaa tagcaagtta aaataaggct agtccgttat caacttgaaa aagtggcacc
     3421 gagtcggtgc ttttttgaat tctagatctt gagacaaatg gcagtattca tccacaacaa
     3481 ctttgtataa taaagttgtt tcccatgatt ccttcatatt tgcatatacg atacaaggct
     3541 gttagagaga taattagaat taatttgact gtaaacacaa agatattagt acaaaatacg
     3601 tgacgtagaa agtaataatt tcttgggtag tttgcagttt taaaattatg ttttaaaatg
     3661 gactatcata tgcttaccgt aacttgaaag tatttcgatt tcttggcttt atatatcttg
     3721 tggaaaggac gaaacaccga ggaggatggc gacgccggtt ttagagctag aaatagcaag
     3781 ttaaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttg
     3841 aattctagat cttgagacaa atggcagtat tcatccacac ccagctttct tgtacaaagt
     3901 tggcattata agaaagcatt gcttatcaat ttgttgcaac gaacaggtca ctatcagtca
     3961 aaataaaatc attatttgcc atccagctga tatcccctat agtgagtcgt attacatggt
     4021 catagctgtt tcctggcagc tctggcccgt gtctcaaaat ctctgatgtt acattgcaca
     4081 agataaaaat atatcatcat gcctcctcta gaccagccag gacagaaatg cctcgacttc
     4141 gctgctaccc aaggttgccg ggtgacgcac accgtggaaa cggatgaagg cacgaaccca
     4201 gtggacataa gcctgttcgg ttcgtaagct gtaatgcaag tagcgtatgc gctcacgcaa
     4261 ctggtccaga accttgaccg aacgcagcgg tggtaacggc gcagtggcgg ttttcatggc
     4321 ttgttatgac tgtttttttg gggtacagtc tatgcctcgg gcatccaagc agcaagcgcg
     4381 ttacgccgtg ggtcgatgtt tgatgttatg gagcagcaac gatgttacgc agcagggcag
     4441 tcgccctaaa acaaagttaa acattatgag ggaagcggtg atcgccgaag tatcgactca
     4501 actatcagag gtagttggcg tcatcgagcg ccatctcgaa ccgacgttgc tggccgtaca
     4561 tttgtacggc tccgcagtgg atggcggcct gaagccacac agtgatattg atttgctggt
     4621 tacggtgacc gtaaggcttg atgaaacaac gcggcgagct ttgatcaacg accttttgga
     4681 aacttcggct tcccctggag agagcgagat tctccgcgct gtagaagtca ccattgttgt
     4741 gcacgacgac atcattccgt ggcgttatcc agctaagcgc gaactgcaat ttggagaatg
     4801 gcagcgcaat gacattcttg caggtatctt cgagccagcc acgatcgaca ttgatctggc
     4861 tatcttgctg acaaaagcaa gagaacatag cgttgccttg gtaggtccag cggcggagga
     4921 actctttgat ccggttcctg aacaggatct atttgaggcg ctaaatgaaa ccttaacgct
     4981 atggaactcg ccgcccgact gggctggcga tgagcgaaat gtagtgctta cgttgtcccg
     5041 catttggtac agcgcagtaa ccggcaaaat cgcgccgaag gatgtcgctg ccgactgggc
     5101 aatggagcgc ctgccggccc agtatcagcc cgtcatactt gaagctagac aggcttatct
     5161 tggacaagaa gaagatcgct tggcctcgcg cgcagatcag ttggaagaat ttgtccacta
     5221 cgtgaaaggc gagatcacca aggtagtcgg caaataaccc tcgagccacc catgaccaaa
     5281 atcccttaac gtgagttacg cgtcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     5341 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     5401 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     5461 aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg
     5521 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     5581 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     5641 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     5701 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     5761 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     5821 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     5881 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     5941 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
     6001 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
     6061 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
     6121 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat g
//
