LOCUS       sequence-263212-        4947 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor vector for gRNA subcloning. Required for gRNA cloning into the
            RCAS-DV. Also constitutively expresses Puromycin linked to TagBFP..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4947)
  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 4947)
  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-PGKpuro2ABFP
FEATURES             Location/Qualifiers
     source          1..4947
                     /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             2299..3000
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     protein_bind    complement(3072..3171)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(3188..3207)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3189..3207)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3212..3228)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3212..3228)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3330..3349)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3341..4150
                     /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(3408..3427)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      4297..4885
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4786..4805
                     /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 gagcgagctg attaaggaga acatgcacat gaagctgtac
     2341 atggagggca ccgtggacaa ccatcacttc aagtgcacat ccgagggcga aggcaagccc
     2401 tacgagggca cccagaccat gagaatcaag gtggtcgagg gcggccctct ccccttcgcc
     2461 ttcgacatcc tggctactag cttcctctac ggcagcaaga ccttcatcaa ccacacccag
     2521 ggcatccccg acttcttcaa gcagtccttc cctgagggct tcacatggga gagagtcacc
     2581 acatacgagg acgggggcgt gctgaccgct acccaggaca ccagcctcca ggacggctgc
     2641 ctcatctaca acgtcaagat cagaggggtg aacttcacat ccaacggccc tgtgatgcag
     2701 aagaaaacac tcggctggga ggccttcacc gagacgctgt accccgctga cggcggcctg
     2761 gaaggcagaa acgacatggc cctgaagctc gtgggcggga gccatctgat cgcaaacatc
     2821 aagaccacat atagatccaa gaaacccgct aagaacctca agatgcctgg cgtctactat
     2881 gtggactaca gactggaaag aatcaaggag gccaacaacg agacctacgt cgagcagcac
     2941 gaggtggcag tggccagata ctgcgacctc cctagcaaac tggggcacaa gcttaattga
     3001 gcggccgcta ggtaccttta agaccaatga cttacaaggc agctgtagat cttagccact
     3061 ttttaaaaga aacccagctt tcttgtacaa agttggcatt ataagaaagc attgcttatc
     3121 aatttgttgc aacgaacagg tcactatcag tcaaaataaa atcattattt gccatccagc
     3181 tgatatcccc tatagtgagt cgtattacat ggtcatagct gtttcctggc agctctggcc
     3241 cgtgtctcaa aatctctgat gttacattgc acaagataaa aatatatcat catgaacaat
     3301 aaaactgtct gcttacataa acagtaatac aaggggtgtt atgagccata ttcaacggga
     3361 aacgtcgagg ccgcgattaa attccaacat ggatgctgat ttatatgggt ataaatgggc
     3421 tcgcgataat gtcgggcaat caggtgcgac aatctatcgc ttgtatggga agcccgatgc
     3481 gccagagttg tttctgaaac atggcaaagg tagcgttgcc aatgatgtta cagatgagat
     3541 ggtcagacta aactggctga cggaatttat gcctcttccg accatcaagc attttatccg
     3601 tactcctgat gatgcatggt tactcaccac tgcgatcccc ggaaaaacag cattccaggt
     3661 attagaagaa tatcctgatt caggtgaaaa tattgttgat gcgctggcag tgttcctgcg
     3721 ccggttgcat tcgattcctg tttgtaattg tccttttaac agcgatcgcg tatttcgtct
     3781 cgctcaggcg caatcacgaa tgaataacgg tttggttgat gcgagtgatt ttgatgacga
     3841 gcgtaatggc tggcctgttg aacaagtctg gaaagaaatg cataaacttt tgccattctc
     3901 accggattca gtcgtcactc atggtgattt ctcacttgat aaccttattt ttgacgaggg
     3961 gaaattaata ggttgtattg atgttggacg agtcggaatc gcagaccgat accaggatct
     4021 tgccatccta tggaactgcc tcggtgagtt ttctccttca ttacagaaac ggctttttca
     4081 aaaatatggt attgataatc ctgatatgaa taaattgcag tttcatttga tgctcgatga
     4141 gtttttctaa tcagaattgg ttaattggtt gtaacactgg cagagcatta cgctgacttg
     4201 acgggacggc gcaagctcat gaccaaaatc ccttaacgtg agttacgcgt cgttccactg
     4261 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     4321 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     4381 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     4441 tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     4501 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     4561 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     4621 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     4681 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     4741 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     4801 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     4861 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     4921 cttttgctgg ccttttgctc acatgtt
//
