LOCUS       sequence-350675-        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"
     protein_bind    1..100
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        160..408
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     160..180
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     331..350
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        425..500
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        523..1022
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(558..581)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     955..974
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             1062..1658
                     /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(1062..1081)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1558..1578
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             1677..1730
                     /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             2457..2483
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     protein_bind    complement(2558..2657)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2674..2693)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2675..2693)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2698..2714)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2698..2714)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2816..2835)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2827..3636
                     /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(2894..2913)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      3783..4371
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4272..4291
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4525..4542
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(4701..4728)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(4820..4906)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     4955..4977
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4969..4986
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4970..4986
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 caaataatga ttttattttg actgatagtg acctgttcgt tgcaacaaat tgatgagcaa
       61 tgctttttta taatgccaac tttgtacaaa aaagcaggct taatccagtt tggttagtac
      121 cgggccctac gcgttactcg agccaaggtc gggcaggaag agggcctatt tcccatgatt
      181 ccttcatatt tgcatatacg atacaaggct gttagagaga taattagaat taatttgact
      241 gtaaacacaa agatattagt acaaaatacg tgacgtagaa agtaataatt tcttgggtag
      301 tttgcagttt taaaattatg ttttaaaatg gactatcata tgcttaccgt aacttgaaag
      361 tatttcgatt tcttggcttt atatatcttg tggaaaggac gaaacaccgg gtcttcgaga
      421 agacgtttta gagctagaaa tagcaagtta aaataaggct agtccgttat caacttgaaa
      481 aagtggcacc gagtcggtgc tttttttgga tccaattcta ccgggtaggg gaggcgcttt
      541 tcccaaggca gtctggagca tgcgctttag cagccccgct gggcacttgg cgctacacaa
      601 gtggcctctg gcctcgcaca cattccacat ccaccggtag gcgccaaccg gctccgttct
      661 ttggtggccc cttcgcgcca ccttctactc ctcccctagt caggaagttc ccccccgccc
      721 cgcagctcgc gtcgtgcagg acgtgacaaa tggaagtagc acgtctcact agtctcgtgc
      781 agatggacag caccgctgag caatggaagc gggtaggcct ttggggcagc ggccaatagc
      841 agctttgctc cttcgctttc tgggctcaga ggctgggaag gggtgggtcc gggggcgggc
      901 tcaggggcgg gctcaggggc ggggcgggcg cccgaaggtc ctccggaggc ccggcattct
      961 gcacgcttca aaagcgcacg tctgccgcgc tgttctcctc ttcctcatct ccgggccttt
     1021 cgacctgcat ccatctagat ctcgagcagc tgaagcttac catgaccgag tacaagccca
     1081 cggtgcgcct cgccacccgc gacgacgtcc ccagggccgt acgcaccctc gccgccgcgt
     1141 tcgccgacta ccccgccacg cgccacaccg tcgatccgga ccgccacatc gagcgggtca
     1201 ccgagctgca agaactcttc ctcacgcgcg tcgggctcga catcggcaag gtgtgggtcg
     1261 cggacgacgg cgccgcggtg gcggtctgga ccacgccgga gagcgtcgaa gcgggggcgg
     1321 tgttcgccga gatcggcccg cgcatggccg agttgagcgg ttcccggctg gccgcgcagc
     1381 aacagatgga aggcctcctg gcgccgcacc ggcccaagga gcccgcgtgg ttcctggcca
     1441 ccgtcggcgt ctcgcccgac caccagggca agggtctggg cagcgccgtc gtgctccccg
     1501 gagtggaggc ggccgagcgc gccggggtgc ccgccttcct ggagacctcc gcgccccgca
     1561 acctcccctt ctacgagcgg ctcggcttca ccgtcaccgc cgacgtcgag gtgcccgaag
     1621 gaccgcgcac ctggtgcatg acccgcaagc ccggtgccgg cggcgggtcc ggaggagagg
     1681 gcagaggaag tctcctaaca tgcggtgacg tggaggagaa tcctggccca atgaatcgct
     1741 gctgggcgct cttcctgtct ctctgctgct acctgcgtct ggtcagcgcc gagggggacc
     1801 ccattcccga ggagctttat gagatgctga gtgaccactc gatccgctcc tttgatgatc
     1861 tccaacgcct gctgcacgga gaccccggag aggaagatgg ggccgagttg gacctgaaca
     1921 tgacccgctc ccactctgga ggcgagctgg agagcttggc tcgtggaaga aggagcctgg
     1981 gttccctgac cattgctgag ccggccatga tcgccgagtg caagacgcgc accgaggtgt
     2041 tcgagatctc ccggcgcctc atagaccgca ccaacgccaa cttcctggtg tggccgccct
     2101 gtgtggaggt gcagcgctgc tccggctgct gcaacaaccg caacgtgcag tgccgcccca
     2161 cccaggtgca gctgcgacct gtccaggtga gaaagatcga gattgtgcgg aagaagccaa
     2221 tctttaagaa ggccacggtg acgctggagg atcacctggc atgcaagtgt gagacagtgg
     2281 cagctgcacg gcctgtgacc cgaagcccgg ggggttccca ggagcagcga gccaaaacgc
     2341 cccaaactcg ggtgaccatt cggacggtgc gagtccgccg gccccccaag ggcaagcacc
     2401 ggaaattcaa gcacacgcat gacaagacgg cactgaagga gacccttgga gccgcctacc
     2461 cttatgacgt gccagattat gcctaggcgg ccgctaggta cctttaagac caatgactta
     2521 caaggcagct gtagatctta gccacttttt aaaagaaacc cagctttctt gtacaaagtt
     2581 ggcattataa gaaagcattg cttatcaatt tgttgcaacg aacaggtcac tatcagtcaa
     2641 aataaaatca ttatttgcca tccagctgat atcccctata gtgagtcgta ttacatggtc
     2701 atagctgttt cctggcagct ctggcccgtg tctcaaaatc tctgatgtta cattgcacaa
     2761 gataaaaata tatcatcatg aacaataaaa ctgtctgctt acataaacag taatacaagg
     2821 ggtgttatga gccatattca acgggaaacg tcgaggccgc gattaaattc caacatggat
     2881 gctgatttat atgggtataa atgggctcgc gataatgtcg ggcaatcagg tgcgacaatc
     2941 tatcgcttgt atgggaagcc cgatgcgcca gagttgtttc tgaaacatgg caaaggtagc
     3001 gttgccaatg atgttacaga tgagatggtc agactaaact ggctgacgga atttatgcct
     3061 cttccgacca tcaagcattt tatccgtact cctgatgatg catggttact caccactgcg
     3121 atccccggaa aaacagcatt ccaggtatta gaagaatatc ctgattcagg tgaaaatatt
     3181 gttgatgcgc tggcagtgtt cctgcgccgg ttgcattcga ttcctgtttg taattgtcct
     3241 tttaacagcg atcgcgtatt tcgtctcgct caggcgcaat cacgaatgaa taacggtttg
     3301 gttgatgcga gtgattttga tgacgagcgt aatggctggc ctgttgaaca agtctggaaa
     3361 gaaatgcata aacttttgcc attctcaccg gattcagtcg tcactcatgg tgatttctca
     3421 cttgataacc ttatttttga cgaggggaaa ttaataggtt gtattgatgt tggacgagtc
     3481 ggaatcgcag accgatacca ggatcttgcc atcctatgga actgcctcgg tgagttttct
     3541 ccttcattac agaaacggct ttttcaaaaa tatggtattg ataatcctga tatgaataaa
     3601 ttgcagtttc atttgatgct cgatgagttt ttctaatcag aattggttaa ttggttgtaa
     3661 cactggcaga gcattacgct gacttgacgg gacggcgcaa gctcatgacc aaaatccctt
     3721 aacgtgagtt acgcgtcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     3781 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     3841 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     3901 ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac
     3961 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     4021 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     4081 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     4141 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     4201 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     4261 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     4321 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     4381 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
     4441 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct
     4501 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca
     4561 atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg
     4621 tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atacgcgtac cgctagccag
     4681 gaagagtttg tagaaacgca aaaaggccat ccgtcaggat ggccttctgc ttagtttgat
     4741 gcctggcagt ttatggcggg cgtcctgccc gccaccctcc gggccgttgc ttcacaacgt
     4801 tcaaatccgc tcccggcgga tttgtcctac tcaggagagc gttcaccgac aaacaacaga
     4861 taaaacgaaa ggcccagttt tccgactgag cctttcgttt tatttgatgc ctggcagttc
     4921 cctactctcg cgttaacgct agcatggatg ttttcccagt cacgacgttg taaaacgacg
     4981 gccagtctta agctcgggcc c
//
