LOCUS       sequence-379405-        4918 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pShuttle.Cre encoding sgRNAs targeting Trp53 and Rb1 genes.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4918)
  AUTHORS   Merle N, Elmshauser S, Strassheimer F, Wanzel M, Konig AM, Funk J, 
            Neumann M, Kochhan K, Helmprobst F, Pagenstecher A, Nist A, 
            Mernberger M, Schneider A, Braun T, Borggrefe T, Savai R, Timofeev 
            O, Stiewe T
  TITLE     Monitoring autochthonous lung tumors induced by somatic CRISPR gene 
            editing in mice using a secreted luciferase.
  JOURNAL   Mol Cancer. 2022 Oct 3;21(1):191. doi: 10.1186/s12943-022-01661-2.
  PUBMED    36192757
REFERENCE   2  (bases 1 to 4918)
  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"; journalName: "Mol 
            Cancer."; date: "2022-10-3"; volume: "21(1):191. doi"; pages: " 
            10.1186/s12943-022-01661-2"
COMMENT     Sequence Label: pShuttle-PR.Cre
FEATURES             Location/Qualifiers
     source          1..4918
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     92..109
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(268..295)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(387..473)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     522..544
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     536..553
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     537..553
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    569..668
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        680..928
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        950..1025
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        1032..1280
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        1301..1376
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        1386..1689
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1690..1893
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1844..1864
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1890..1914
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             1982..2002
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2003..3031
                     /codon_start=1
                     /product="site-specific recombinase"
                     /label=Cre
                     /note="Cre is a site-specific recombinase from 
                     bacteriophage P1. Recombination occurs at loxP sequences."
                     /translation="SNLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKMLQ
                     SVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGLP
                     RPSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAFL
                     GIAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVER
                     WISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQR
                     YLAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDSETGAMVRLLED
                     GD"
     protein_bind    complement(3043..3142)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(3159..3178)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3160..3178)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3183..3199)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3183..3199)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3301..3320)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3312..4121
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(3379..3398)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      4268..4856
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4757..4776
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
       61 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
      121 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
      181 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaata cgcgtaccgc
      241 tagccaggaa gagtttgtag aaacgcaaaa aggccatccg tcaggatggc cttctgctta
      301 gtttgatgcc tggcagttta tggcgggcgt cctgcccgcc accctccggg ccgttgcttc
      361 acaacgttca aatccgctcc cggcggattt gtcctactca ggagagcgtt caccgacaaa
      421 caacagataa aacgaaaggc ccagtcttcc gactgagcct ttcgttttat ttgatgcctg
      481 gcagttccct actctcgcgt taacgctagc atggatgttt tcccagtcac gacgttgtaa
      541 aacgacggcc agtcttaagc tcgggcccca aataatgatt ttattttgac tgatagtgac
      601 ctgttcgttg caacaaattg atgagcaatg cttttttata atgccaactt tgtacaaaaa
      661 agcaggcacc atggaggctg agggcctatt tcccatgatt ccttcatatt tgcatatacg
      721 atacaaggct gttagagaga taattggaat taatttgact gtaaacacaa agatattagt
      781 acaaaatacg tgacgtagaa agtaataatt tcttgggtag tttgcagttt taaaattatg
      841 ttttaaaatg gactatcata tgcttaccgt aacttgaaag tatttcgatt tcttggcttt
      901 atatatcttg tggaaaggac gaaacaccgc ataaggtacc accacgctgg ttttagagct
      961 agaaatagca agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc
     1021 ggtgcttttt tgagggccta tttcccatga ttccttcata tttgcatata cgatacaagg
     1081 ctgttagaga gataattgga attaatttga ctgtaaacac aaagatatta gtacaaaata
     1141 cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta tgttttaaaa
     1201 tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct ttatatatct
     1261 tgtggaaagg acgaaacacc gaacagattt gtccttcccg gttttagagc tagaaatagc
     1321 aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt
     1381 ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc
     1441 ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac tttccattga
     1501 cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat
     1561 atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc
     1621 cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct
     1681 attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca
     1741 cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat
     1801 caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg
     1861 cgtgtacggt gggaggtcta tataagcaga gctcgtttag tgaaccgtca gatcgcctgg
     1921 agacgccatc cacgctgttt tgacctccat agaagacacc gactctagag gatctaccat
     1981 gcccaagaag aagaggaagg tgtccaattt actgaccgta caccaaaatt tgcctgcatt
     2041 accggtcgat gcaacgagtg atgaggttcg caagaacctg atggacatgt tcagggatcg
     2101 ccaggcgttt tctgagcata cctggaaaat gcttcagtcc gtttgccggt cgtgggcggc
     2161 atggtgcaag ttgaataacc ggaaatggtt tcccgcagaa cctgaagatg ttcgcgatta
     2221 tcttctatat cttcaggcgc gcggtctggc agtaaaaact atccagcaac atttgggcca
     2281 gctaaacatg cttcatcgtc ggtccgggct gccacgacca agtgacagca atgctgtttc
     2341 actggttatg cggcggatcc gaaaagaaaa cgttgatgcc ggtgaacgtg caaaacaggc
     2401 tctagcgttc gaacgcactg atttcgacca ggttcgttca ctcatggaaa atagcgatcg
     2461 ctgccaggat atacgtaatc tggcatttct ggggattgct tataacaccc tgttacgtat
     2521 agccgaaatt gccaggatca gggttaaaga tatctcacgt actgacggtg ggagaatgtt
     2581 aatccatatt ggcagaacga aaacgctggt tagcaccgca ggtgtagaga aggcacttag
     2641 cctgggggta actaaactgg tcgagcgatg gatttccgtc tctggtgtag ctgatgatcc
     2701 gaataactac ctgttttgcc gggtcagaaa aaatggtgtt gccgcgccat ctgccaccag
     2761 ccagctatca actcgcgccc tggaagggat ttttgaagca actcatcgat tgatttacgg
     2821 cgctaaggat gactctggtc agagatacct ggcctggtct ggacacagtg cccgtgtcgg
     2881 agccgcgcga gatatggccc gcgctggagt ttcaataccg gagatcatgc aagctggtgg
     2941 ctggaccaat gtaaatattg tcatgaacta tatccgtaac ctggatagtg aaacaggggc
     3001 aatggtgcgc ctgctggaag atggcgatta gggaattcta gaacccagct ttcttgtaca
     3061 aagttggcat tataagaaag cattgcttat caatttgttg caacgaacag gtcactatca
     3121 gtcaaaataa aatcattatt tgccatccag ctgatatccc ctatagtgag tcgtattaca
     3181 tggtcatagc tgtttcctgg cagctctggc ccgtgtctca aaatctctga tgttacattg
     3241 cacaagataa aaatatatca tcatgaacaa taaaactgtc tgcttacata aacagtaata
     3301 caaggggtgt tatgagccat attcaacggg aaacgtcgag gccgcgatta aattccaaca
     3361 tggatgctga tttatatggg tataaatggg ctcgcgataa tgtcgggcaa tcaggtgcga
     3421 caatctatcg cttgtatggg aagcccgatg cgccagagtt gtttctgaaa catggcaaag
     3481 gtagcgttgc caatgatgtt acagatgaga tggtcagact aaactggctg acggaattta
     3541 tgcctcttcc gaccatcaag cattttatcc gtactcctga tgatgcatgg ttactcacca
     3601 ctgcgatccc cggaaaaaca gcattccagg tattagaaga atatcctgat tcaggtgaaa
     3661 atattgttga tgcgctggca gtgttcctgc gccggttgca ttcgattcct gtttgtaatt
     3721 gtccttttaa cagcgatcgc gtatttcgtc tcgctcaggc gcaatcacga atgaataacg
     3781 gtttggttga tgcgagtgat tttgatgacg agcgtaatgg ctggcctgtt gaacaagtct
     3841 ggaaagaaat gcataaactt ttgccattct caccggattc agtcgtcact catggtgatt
     3901 tctcacttga taaccttatt tttgacgagg ggaaattaat aggttgtatt gatgttggac
     3961 gagtcggaat cgcagaccga taccaggatc ttgccatcct atggaactgc ctcggtgagt
     4021 tttctccttc attacagaaa cggctttttc aaaaatatgg tattgataat cctgatatga
     4081 ataaattgca gtttcatttg atgctcgatg agtttttcta atcagaattg gttaattggt
     4141 tgtaacactg gcagagcatt acgctgactt gacgggacgg cgcaagctca tgaccaaaat
     4201 cccttaacgt gagttacgcg tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     4261 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     4321 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     4381 ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc
     4441 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     4501 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     4561 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     4621 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     4681 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     4741 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     4801 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     4861 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgtt
//
