LOCUS       sequence-263217-        5001 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor vector for gRNA subcloning. Required for gRNA cloning into the
            RCAS-DV. Also constitutively expresses PDGFB..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5001)
  AUTHORS   Oldrini B, Curiel-Garcia A, Marques C, Matia V, Uluckan O, 
            Grana-Castro O, Torres-Ruiz R, Rodriguez-Perales S, Huse JT, 
            Squatrito M
  TITLE     Somatic genome editing with the RCAS-TVA-CRISPR-Cas9 system for 
            precision tumor modeling.
  JOURNAL   Nat Commun. 2018 Apr 13;9(1):1466. doi: 10.1038/s41467-018-03731-w.
  PUBMED    29654229
REFERENCE   2  (bases 1 to 5001)
  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: "Nat 
            Commun."; date: "2018-04-13"; volume: "9(1):1466. doi"; pages: " 
            10.1038/s41467-018-03731-w"
COMMENT     Sequence Label: pDONR-U6gRNA-PGKpuro2APDGFB
FEATURES             Location/Qualifiers
     source          1..5001
                     /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        728..976
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     728..748
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     899..918
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        993..1068
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        1091..1590
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(1126..1149)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     1523..1542
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             1630..2226
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(1630..1649)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2126..2146
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             2245..2298
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             3025..3051
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     protein_bind    complement(3126..3225)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(3242..3261)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3243..3261)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3266..3282)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3266..3282)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3384..3403)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3395..4204
                     /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(3462..3481)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      4351..4939
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4840..4859
                     /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 ccagttttcc 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 agcaggctta atccagtttg gttagtaccg ggccctacgc gttactcgag ccaaggtcgg
      721 gcaggaagag ggcctatttc ccatgattcc ttcatatttg catatacgat acaaggctgt
      781 tagagagata attagaatta atttgactgt aaacacaaag atattagtac aaaatacgtg
      841 acgtagaaag taataatttc ttgggtagtt tgcagtttta aaattatgtt ttaaaatgga
      901 ctatcatatg cttaccgtaa cttgaaagta tttcgatttc ttggctttat atatcttgtg
      961 gaaaggacga aacaccgggt cttcgagaag acgttttaga gctagaaata gcaagttaaa
     1021 ataaggctag tccgttatca acttgaaaaa gtggcaccga gtcggtgctt tttttggatc
     1081 caattctacc gggtagggga ggcgcttttc ccaaggcagt ctggagcatg cgctttagca
     1141 gccccgctgg gcacttggcg ctacacaagt ggcctctggc ctcgcacaca ttccacatcc
     1201 accggtaggc gccaaccggc tccgttcttt ggtggcccct tcgcgccacc ttctactcct
     1261 cccctagtca ggaagttccc ccccgccccg cagctcgcgt cgtgcaggac gtgacaaatg
     1321 gaagtagcac gtctcactag tctcgtgcag atggacagca ccgctgagca atggaagcgg
     1381 gtaggccttt ggggcagcgg ccaatagcag ctttgctcct tcgctttctg ggctcagagg
     1441 ctgggaaggg gtgggtccgg gggcgggctc aggggcgggc tcaggggcgg ggcgggcgcc
     1501 cgaaggtcct ccggaggccc ggcattctgc acgcttcaaa agcgcacgtc tgccgcgctg
     1561 ttctcctctt cctcatctcc gggcctttcg acctgcatcc atctagatct cgagcagctg
     1621 aagcttacca tgaccgagta caagcccacg gtgcgcctcg ccacccgcga cgacgtcccc
     1681 agggccgtac gcaccctcgc cgccgcgttc gccgactacc ccgccacgcg ccacaccgtc
     1741 gatccggacc gccacatcga gcgggtcacc gagctgcaag aactcttcct cacgcgcgtc
     1801 gggctcgaca tcggcaaggt gtgggtcgcg gacgacggcg ccgcggtggc ggtctggacc
     1861 acgccggaga gcgtcgaagc gggggcggtg ttcgccgaga tcggcccgcg catggccgag
     1921 ttgagcggtt cccggctggc cgcgcagcaa cagatggaag gcctcctggc gccgcaccgg
     1981 cccaaggagc ccgcgtggtt cctggccacc gtcggcgtct cgcccgacca ccagggcaag
     2041 ggtctgggca gcgccgtcgt gctccccgga gtggaggcgg ccgagcgcgc cggggtgccc
     2101 gccttcctgg agacctccgc gccccgcaac ctccccttct acgagcggct cggcttcacc
     2161 gtcaccgccg acgtcgaggt gcccgaagga ccgcgcacct ggtgcatgac ccgcaagccc
     2221 ggtgccggcg gcgggtccgg aggagagggc agaggaagtc tcctaacatg cggtgacgtg
     2281 gaggagaatc ctggcccaat gaatcgctgc tgggcgctct tcctgtctct ctgctgctac
     2341 ctgcgtctgg tcagcgccga gggggacccc attcccgagg agctttatga gatgctgagt
     2401 gaccactcga tccgctcctt tgatgatctc caacgcctgc tgcacggaga ccccggagag
     2461 gaagatgggg ccgagttgga cctgaacatg acccgctccc actctggagg cgagctggag
     2521 agcttggctc gtggaagaag gagcctgggt tccctgacca ttgctgagcc ggccatgatc
     2581 gccgagtgca agacgcgcac cgaggtgttc gagatctccc ggcgcctcat agaccgcacc
     2641 aacgccaact tcctggtgtg gccgccctgt gtggaggtgc agcgctgctc cggctgctgc
     2701 aacaaccgca acgtgcagtg ccgccccacc caggtgcagc tgcgacctgt ccaggtgaga
     2761 aagatcgaga ttgtgcggaa gaagccaatc tttaagaagg ccacggtgac gctggaggat
     2821 cacctggcat gcaagtgtga gacagtggca gctgcacggc ctgtgacccg aagcccgggg
     2881 ggttcccagg agcagcgagc caaaacgccc caaactcggg tgaccattcg gacggtgcga
     2941 gtccgccggc cccccaaggg caagcaccgg aaattcaagc acacgcatga caagacggca
     3001 ctgaaggaga cccttggagc cgcctaccct tatgacgtgc cagattatgc ctaggcggcc
     3061 gctaggtacc tttaagacca atgacttaca aggcagctgt agatcttagc cactttttaa
     3121 aagaaaccca gctttcttgt acaaagttgg cattataaga aagcattgct tatcaatttg
     3181 ttgcaacgaa caggtcacta tcagtcaaaa taaaatcatt atttgccatc cagctgatat
     3241 cccctatagt gagtcgtatt acatggtcat agctgtttcc tggcagctct ggcccgtgtc
     3301 tcaaaatctc tgatgttaca ttgcacaaga taaaaatata tcatcatgaa caataaaact
     3361 gtctgcttac ataaacagta atacaagggg tgttatgagc catattcaac gggaaacgtc
     3421 gaggccgcga ttaaattcca acatggatgc tgatttatat gggtataaat gggctcgcga
     3481 taatgtcggg caatcaggtg cgacaatcta tcgcttgtat gggaagcccg atgcgccaga
     3541 gttgtttctg aaacatggca aaggtagcgt tgccaatgat gttacagatg agatggtcag
     3601 actaaactgg ctgacggaat ttatgcctct tccgaccatc aagcatttta tccgtactcc
     3661 tgatgatgca tggttactca ccactgcgat ccccggaaaa acagcattcc aggtattaga
     3721 agaatatcct gattcaggtg aaaatattgt tgatgcgctg gcagtgttcc tgcgccggtt
     3781 gcattcgatt cctgtttgta attgtccttt taacagcgat cgcgtatttc gtctcgctca
     3841 ggcgcaatca cgaatgaata acggtttggt tgatgcgagt gattttgatg acgagcgtaa
     3901 tggctggcct gttgaacaag tctggaaaga aatgcataaa cttttgccat tctcaccgga
     3961 ttcagtcgtc actcatggtg atttctcact tgataacctt atttttgacg aggggaaatt
     4021 aataggttgt attgatgttg gacgagtcgg aatcgcagac cgataccagg atcttgccat
     4081 cctatggaac tgcctcggtg agttttctcc ttcattacag aaacggcttt ttcaaaaata
     4141 tggtattgat aatcctgata tgaataaatt gcagtttcat ttgatgctcg atgagttttt
     4201 ctaatcagaa ttggttaatt ggttgtaaca ctggcagagc attacgctga cttgacggga
     4261 cggcgcaagc tcatgaccaa aatcccttaa cgtgagttac gcgtcgttcc actgagcgtc
     4321 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     4381 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     4441 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct
     4501 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     4561 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     4621 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     4681 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     4741 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     4801 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     4861 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     4921 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     4981 ctggcctttt gctcacatgt t
//
