LOCUS       sequence-237367-        3716 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pMAGIC L5-L4 entry plasmid, contains empty human U6-driven SaCas9 
            gRNA scaffold and CMV promoter for 4-component MultiSite Gateway Pro
            assembly. Protospacer motif can be inserted after BsaI digestion.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3716)
  AUTHORS   Haldeman JM, Conway AE, Arlotto ME, Slentz DH, Muoio DM, Becker TC, 
            Newgard CB
  TITLE     Creation of versatile cloning platforms for transgene expression and
            dCas9-based epigenome editing.
  JOURNAL   Nucleic Acids Res. 2018 Dec 27. pii: 5264291. doi: 
            10.1093/nar/gky1286.
  PUBMED    30590691
REFERENCE   2  (bases 1 to 3716)
  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: "Nucleic 
            Acids Res. 2018 Dec 27. pii: 5264291. doi: 10.1093/nar/gky1286."
COMMENT     Sequence Label: KB701: pMAGIC (L5-L4) hU6::SaCas9 gRNA scaffol...
FEATURES             Location/Qualifiers
     source          1..3716
                     /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    570..665
                     /gene="mutant version of attL"
                     /label=attL5
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        674..922
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     674..694
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     845..864
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     enhancer        1213..1592
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1593..1796
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1747..1767
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1793..1817
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    complement(1831..1926)
                     /gene="mutant version of attL"
                     /label=attL4
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1957..1976)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1958..1976)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1981..1997)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1981..1997)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2099..2118)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2110..2919
                     /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(2177..2196)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      3066..3654
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3555..3574
                     /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 ccagtcttcc gactgagcct ttcgttttat ttgatgcctg
      481 gcagttccct actctcgcgt taacgctagc atggatgttt tcccagtcac gacgttgtaa
      541 aacgacggcc agtcttaagc tcgggcccca aataatgatt ttattttgac tgatagtgac
      601 ctgttcgttg caacaaattg atgagcaatg cttttttata atgccaactt tgtatacaaa
      661 agttgtggta cctgagggcc tatttcccat gattccttca tatttgcata tacgatacaa
      721 ggctgttaga gagataattg gaattaattt gactgtaaac acaaagatat tagtacaaaa
      781 tacgtgacgt agaaagtaat aatttcttgg gtagtttgca gttttaaaat tatgttttaa
      841 aatggactat catatgctta ccgtaacttg aaagtatttc gatttcttgg ctttatatat
      901 cttgtggaaa ggacgaaaca ccggagacca cggcaggtct cagtttaagt actctgtgct
      961 ggaaacagca cagaatctac taaacaaggc aaaatgccgt gtttatctcg tcaacttgtt
     1021 ggcgagattt ttgcggccgc tgtacatacg actctagaac gatctataca ttgaatcaat
     1081 attggcaatt agccatatta gtcattggtt atatagcata aatcaatatt ggctattggc
     1141 cattgcatac gttgtatcta tatcataata tgtacattta tattggctca tgtccaatat
     1201 gaccgccatg ttgacattga ttattgacta gttattaata gtaatcaatt acggggtcat
     1261 tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg
     1321 gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa
     1381 cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact
     1441 tggcagtaca tcaagtgtat catatgccaa gtccgccccc tattgacgtc aatgacggta
     1501 aatggcccgc ctggcattat gcccagtaca tgaccttacg ggactttcct acttggcagt
     1561 acatctacgt attagtcatc gctattacca tggtgatgcg gttttggcag tacaccaatg
     1621 ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg
     1681 ggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaat aaccccgccc
     1741 cgttgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctcgtt
     1801 tagtgaaccg tcagatccgg gaattccacc caacttttct atacaaagtt ggcattataa
     1861 gaaagcattg cttatcaatt tgttgcaacg aacaggtcac tatcagtcaa aataaaatca
     1921 ttatttgcca tccagctgca gggcggccgc gatatcccct atagtgagtc gtattacatg
     1981 gtcatagctg tttcctggca gctctggccc gtgtctcaaa atctctgatg ttacattgca
     2041 caagataaaa atatatcatc atgaacaata aaactgtctg cttacataaa cagtaataca
     2101 aggggtgtta tgagccatat tcaacgggaa acgtcgaggc cgcgattaaa ttccaacatg
     2161 gatgctgatt tatatgggta taaatgggct cgcgataatg tcgggcaatc aggtgcgaca
     2221 atctatcgct tgtatgggaa gcccgatgcg ccagagttgt ttctgaaaca tggcaaaggt
     2281 agcgttgcca atgatgttac agatgagatg gtcagactaa actggctgac ggaatttatg
     2341 cctcttccga ccatcaagca ttttatccgt actcctgatg atgcatggtt actcaccact
     2401 gcgatccccg gaaaaacagc attccaggta ttagaagaat atcctgattc aggtgaaaat
     2461 attgttgatg cgctggcagt gttcctgcgc cggttgcatt cgattcctgt ttgtaattgt
     2521 ccttttaaca gcgatcgcgt atttcgtctc gctcaggcgc aatcacgaat gaataacggt
     2581 ttggttgatg cgagtgattt tgatgacgag cgtaatggct ggcctgttga acaagtctgg
     2641 aaagaaatgc ataaactttt gccattctca ccggattcag tcgtcactca tggtgatttc
     2701 tcacttgata accttatttt tgacgagggg aaattaatag gttgtattga tgttggacga
     2761 gtcggaatcg cagaccgata ccaggatctt gccatcctat ggaactgcct cggtgagttt
     2821 tctccttcat tacagaaacg gctttttcaa aaatatggta ttgataatcc tgatatgaat
     2881 aaattgcagt ttcatttgat gctcgatgag tttttctaat cagaattggt taattggttg
     2941 taacactggc agagcattac gctgacttga cgggacggcg caagctcatg accaaaatcc
     3001 cttaacgtga gttacgcgtc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     3061 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     3121 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     3181 ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac
     3241 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     3301 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     3361 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     3421 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     3481 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     3541 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     3601 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     3661 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgtt
//
