LOCUS       sequence-406418-        2271 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  ENTR Vector 2 for MultiSite Gateway assembly. Encodes hU6 hACTB 
            sgRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2271)
  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 2271)
  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 ACTB sgRNA
FEATURES             Location/Qualifiers
     source          1..2271
                     /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    1085..1209
                     /gene="mutant version of attR"
                     /label=attR4
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1305..1324)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1316..2125
                     /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(1383..1402)
                     /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 attggaatta atttgactgt aaacacaaag atattagtac aaaatacgtg acgtagaaag
      841 taataatttc ttgggtagtt tgcagtttta aaattatgtt ttaaaatgga ctatcatatg
      901 cttaccgtaa cttgaaagta tttcgatttc ttggctttat atatcttgtg gaaaggacga
      961 aacaccgaca gctccccaca caccacgttt tagagctaga aatagcaagt taaaataagg
     1021 ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gcttttttta tccctaggcc
     1081 cgggccaact ttgtatagaa aagttgaacg agaaacgtaa aatgatataa atatcaatat
     1141 attaaattag attttgcata aaaaacagac tacataatac tgtaaaacac aacatatcca
     1201 gtcactatgc tggcccgtgt ctcaaaatct ctgatgttac attgcacaag ataaaaatat
     1261 atcatcatga acaataaaac tgtctgctta cataaacagt aatacaaggg gtgttatgag
     1321 ccatattcaa cgggaaacgt cgaggccgcg attaaattcc aacatggatg ctgatttata
     1381 tgggtataaa tgggctcgcg ataatgtcgg gcaatcaggt gcgacaatct atcgcttgta
     1441 tgggaagccc gatgcgccag agttgtttct gaaacatggc aaaggtagcg ttgccaatga
     1501 tgttacagat gagatggtca gactaaactg gctgacggaa tttatgcctc ttccgaccat
     1561 caagcatttt atccgtactc ctgatgatgc atggttactc accactgcga tccccggaaa
     1621 aacagcattc caggtattag aagaatatcc tgattcaggt gaaaatattg ttgatgcgct
     1681 ggcagtgttc ctgcgccggt tgcattcgat tcctgtttgt aattgtcctt ttaacagcga
     1741 tcgcgtattt cgtctcgctc aggcgcaatc acgaatgaat aacggtttgg ttgatgcgag
     1801 tgattttgat gacgagcgta atggctggcc tgttgaacaa gtctggaaag aaatgcataa
     1861 acttttgcca ttctcaccgg attcagtcgt cactcatggt gatttctcac ttgataacct
     1921 tatttttgac gaggggaaat taataggttg tattgatgtt ggacgagtcg gaatcgcaga
     1981 ccgataccag gatcttgcca tcctatggaa ctgcctcggt gagttttctc cttcattaca
     2041 gaaacggctt tttcaaaaat atggtattga taatcctgat atgaataaat tgcagtttca
     2101 tttgatgctc gatgagtttt tctaatcaga attggttaat tggttgtaac actggcagag
     2161 cattacgctg acttgacggg acggcgcaag ctcatgacca aaatccctta acgtgagtta
     2221 cgcgtcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt c
//
