LOCUS       sequence-406648-        2317 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  ENTR Vector 2 for MultiSite Gateway assembly. Encodes HEK3 PegRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2317)
  AUTHORS   Aulicino F, Pelosse M, Toelzer C, Capin J, Ilegems E, Meysami P, 
            Rollarson R, Berggren PO, Dillingham MS, Schaffitzel C, Saleem MA, 
            Welsh GI, Berger I
  TITLE     Highly efficient CRISPR-mediated large DNA docking and multiplexed 
            prime editing using a single baculovirus.
  JOURNAL   Nucleic Acids Res. 2022 Jul 22;50(13):7783-7799. doi: 
            10.1093/nar/gkac587.
  PUBMED    35801912
REFERENCE   2  (bases 1 to 2317)
  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"; doi: "10"; journalName: 
            "Nucleic Acids Res"; date: "2022-07-22- 22"; volume: "50"; issue: 
            "13"; pages: "7783-7799"
COMMENT     Sequence Label: pMMK ENTR 2 hU6 HEK3 PegRNA
FEATURES             Location/Qualifiers
     source          1..2317
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    605..700
                     /gene="mutant version of attL"
                     /label=attL3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        718..966
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     718..738
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     889..908
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        987..1062
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    1131..1255
                     /gene="mutant version of attR"
                     /label=attR4
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1351..1370)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1362..2171
                     /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(1429..1448)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcgaaataa tgattttatt ttgactgata gtgacctgtt cgttgcaaca aattgatgag
      661 caatgctttt ttataatgcc aactttgtat aataaagttg gtttaaacga tatcaccgag
      721 ggcctatttc ccatgattcc ttcatatttg catatacgat acaaggctgt tagagagata
      781 attagaatta atttgactgt aaacacaaag atattagtac aaaatacgtg acgtagaaag
      841 taataatttc ttgggtagtt tgcagtttta aaattatgtt ttaaaatgga ctatcatatg
      901 cttaccgtaa cttgaaagta tttcgatttc ttggctttat atatcttgtg gaaaggacga
      961 aacaccggcc cagactgagc acgtgagttt tagagctaga aatagcaagt taaaataagg
     1021 ctagtccgtt atcaacttga aaaagtggga ccgagtcggt cctctgccat caaagcgtgc
     1081 tcagtctgtt tttttaagct tgggccgctc gaggtatccc taggcccggg ccaactttgt
     1141 atagaaaagt tgaacgagaa acgtaaaatg atataaatat caatatatta aattagattt
     1201 tgcataaaaa acagactaca taatactgta aaacacaaca tatccagtca ctatgctggc
     1261 ccgtgtctca aaatctctga tgttacattg cacaagataa aaatatatca tcatgaacaa
     1321 taaaactgtc tgcttacata aacagtaata caaggggtgt tatgagccat attcaacggg
     1381 aaacgtcgag gccgcgatta aattccaaca tggatgctga tttatatggg tataaatggg
     1441 ctcgcgataa tgtcgggcaa tcaggtgcga caatctatcg cttgtatggg aagcccgatg
     1501 cgccagagtt gtttctgaaa catggcaaag gtagcgttgc caatgatgtt acagatgaga
     1561 tggtcagact aaactggctg acggaattta tgcctcttcc gaccatcaag cattttatcc
     1621 gtactcctga tgatgcatgg ttactcacca ctgcgatccc cggaaaaaca gcattccagg
     1681 tattagaaga atatcctgat tcaggtgaaa atattgttga tgcgctggca gtgttcctgc
     1741 gccggttgca ttcgattcct gtttgtaatt gtccttttaa cagcgatcgc gtatttcgtc
     1801 tcgctcaggc gcaatcacga atgaataacg gtttggttga tgcgagtgat tttgatgacg
     1861 agcgtaatgg ctggcctgtt gaacaagtct ggaaagaaat gcataaactt ttgccattct
     1921 caccggattc agtcgtcact catggtgatt tctcacttga taaccttatt tttgacgagg
     1981 ggaaattaat aggttgtatt gatgttggac gagtcggaat cgcagaccga taccaggatc
     2041 ttgccatcct atggaactgc ctcggtgagt tttctccttc attacagaaa cggctttttc
     2101 aaaaatatgg tattgataat cctgatatga ataaattgca gtttcatttg atgctcgatg
     2161 agtttttcta atcagaattg gttaattggt tgtaacactg gcagagcatt acgctgactt
     2221 gacgggacgg cgcaagctca tgaccaaaat cccttaacgt gagttacgcg tcgttccact
     2281 gagcgtcaga ccccgtagaa aagatcaaag gatcttc
//
