LOCUS       sequence-379406-        5270 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pShuttle.Cre encoding sgRNAs targeting Trp53, Rb1 and Rbl2 genes.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5270)
  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 5270)
  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-PRL.Cre
FEATURES             Location/Qualifiers
     source          1..5270
                     /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"
     promoter        1383..1631
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        1653..1728
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        1738..2041
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2042..2245
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2196..2216
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2242..2266
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             2334..2354
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2355..3383
                     /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(3395..3494)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(3511..3530)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3512..3530)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3535..3551)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3535..3551)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3653..3672)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3664..4473
                     /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(3731..3750)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      4620..5208
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5109..5128
                     /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 ttgagggcct atttcccatg attccttcat atttgcatat acgatacaag gctgttagag
     1441 agataattgg aattaatttg actgtaaaca caaagatatt agtacaaaat acgtgacgta
     1501 gaaagtaata atttcttggg tagtttgcag ttttaaaatt atgttttaaa atggactatc
     1561 atatgcttac cgtaacttga aagtatttcg atttcttggc tttatatatc ttgtggaaag
     1621 gacgaaacac cgcccgtgag tcgagttggt gtgttttaga gctagaaata gcaagttaaa
     1681 ataaggctag tccgttatca acttgaaaaa gtggcaccga gtcggtgctt ttttccgcgt
     1741 tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac
     1801 gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg
     1861 ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag
     1921 tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat
     1981 gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat
     2041 ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact cacggggatt
     2101 tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa atcaacggga
     2161 ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta ggcgtgtacg
     2221 gtgggaggtc tatataagca gagctcgttt agtgaaccgt cagatcgcct ggagacgcca
     2281 tccacgctgt tttgacctcc atagaagaca ccgactctag aggatctacc atgcccaaga
     2341 agaagaggaa ggtgtccaat ttactgaccg tacaccaaaa tttgcctgca ttaccggtcg
     2401 atgcaacgag tgatgaggtt cgcaagaacc tgatggacat gttcagggat cgccaggcgt
     2461 tttctgagca tacctggaaa atgcttcagt ccgtttgccg gtcgtgggcg gcatggtgta
     2521 agttgaataa ccggaaatgg tttcccgcag aacctgaaga tgttcgcgat tatcttctat
     2581 atcttcaggc gcgcggtctg gcagtaaaaa ctatccagca acatttgggc cagctaaaca
     2641 tgcttcatcg tcggtccggg ctgccacgac caagtgacag caatgctgtt tcactggtta
     2701 tgcggcggat ccgaaaagaa aacgttgatg ccggtgaacg tgcaaaacag gctctagcgt
     2761 tcgaacgcac tgatttcgac caggttcgtt cactcatgga aaatagcgat cgctgccagg
     2821 atatacgtaa tctggcattt ctggggattg cttataacac cctgttacgt atagccgaaa
     2881 ttgccaggat cagggttaaa gatatctcac gtactgacgg tgggagaatg ttaatccata
     2941 ttggcagaac gaaaacgctg gttagcaccg caggtgtaga gaaggcactt agcctggggg
     3001 taactaaact ggtcgagcga tggatttccg tctctggtgt agctgatgat ccgaataact
     3061 acctgttttg ccgggtcaga aaaaatggtg ttgccgcgcc atctgccacc agccagctat
     3121 caactcgcgc cctggaaggg atttttgaag caactcatcg attgatttac ggcgctaagg
     3181 atgactctgg tcagagatac ctggcctggt ctggacacag tgcccgtgtc ggagccgcgc
     3241 gagatatggc ccgcgctgga gtttcaatac cggagatcat gcaagctggt ggctggacca
     3301 atgtaaatat tgtcatgaac tatatccgta acctggatag tgaaacaggg gcaatggtgc
     3361 gcctgctgga agatggcgat tagggaattc tagaacccag ctttcttgta caaagttggc
     3421 attataagaa agcattgctt atcaatttgt tgcaacgaac aggtcactat cagtcaaaat
     3481 aaaatcatta tttgccatcc agctgatatc ccctatagtg agtcgtatta catggtcata
     3541 gctgtttcct ggcagctctg gcccgtgtct caaaatctct gatgttacat tgcacaagat
     3601 aaaaatatat catcatgaac aataaaactg tctgcttaca taaacagtaa tacaaggggt
     3661 gttatgagcc atattcaacg ggaaacgtcg aggccgcgat taaattccaa catggatgct
     3721 gatttatatg ggtataaatg ggctcgcgat aatgtcgggc aatcaggtgc gacaatctat
     3781 cgcttgtatg ggaagcccga tgcgccagag ttgtttctga aacatggcaa aggtagcgtt
     3841 gccaatgatg ttacagatga gatggtcaga ctaaactggc tgacggaatt tatgcctctt
     3901 ccgaccatca agcattttat ccgtactcct gatgatgcat ggttactcac cactgcgatc
     3961 cccggaaaaa cagcattcca ggtattagaa gaatatcctg attcaggtga aaatattgtt
     4021 gatgcgctgg cagtgttcct gcgccggttg cattcgattc ctgtttgtaa ttgtcctttt
     4081 aacagcgatc gcgtatttcg tctcgctcag gcgcaatcac gaatgaataa cggtttggtt
     4141 gatgcgagtg attttgatga cgagcgtaat ggctggcctg ttgaacaagt ctggaaagaa
     4201 atgcataaac ttttgccatt ctcaccggat tcagtcgtca ctcatggtga tttctcactt
     4261 gataacctta tttttgacga ggggaaatta ataggttgta ttgatgttgg acgagtcgga
     4321 atcgcagacc gataccagga tcttgccatc ctatggaact gcctcggtga gttttctcct
     4381 tcattacaga aacggctttt tcaaaaatat ggtattgata atcctgatat gaataaattg
     4441 cagtttcatt tgatgctcga tgagtttttc taatcagaat tggttaattg gttgtaacac
     4501 tggcagagca ttacgctgac ttgacgggac ggcgcaagct catgaccaaa atcccttaac
     4561 gtgagttacg cgtcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     4621 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     4681 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     4741 agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc
     4801 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     4861 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     4921 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     4981 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     5041 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     5101 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     5161 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     5221 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
//
