LOCUS       sequence-237361-        4233 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pMAGIC L1-R5 entry plasmid, contains empty human U6-driven SaCas9 
            gRNA scaffold and EF1a 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 4233)
  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 4233)
  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: KZ801: pMAGIC (L1-R5) hU6::SaCas9 gRNA scaffol...
FEATURES             Location/Qualifiers
     source          1..4233
                     /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        679..927
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     679..699
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     850..869
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     promoter        1069..2247
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          1300..2238
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     2195..2215
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     promoter        2264..2282
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2296..2419
                     /gene="mutant version of attR"
                     /label=attR5
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        complement(2475..2493)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2498..2514)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2498..2514)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2616..2635)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2627..3436
                     /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(2694..2713)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      3583..4171
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4072..4091
                     /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 tgtacaaaaa
      661 agcaggctta tggtacctga gggcctattt cccatgattc cttcatattt gcatatacga
      721 tacaaggctg ttagagagat aattggaatt aatttgactg taaacacaaa gatattagta
      781 caaaatacgt gacgtagaaa gtaataattt cttgggtagt ttgcagtttt aaaattatgt
      841 tttaaaatgg actatcatat gcttaccgta acttgaaagt atttcgattt cttggcttta
      901 tatatcttgt ggaaaggacg aaacaccgga gaccacggca ggtctcagtt taagtactct
      961 gtgctggaaa cagcacagaa tctactaaac aaggcaaaat gccgtgttta tctcgtcaac
     1021 ttgttggcga gatttttgcg gccgctgtac atacgactct agacgtgagg ctccggtgcc
     1081 cgtcagtggg cagagcgcac atcgcccaca gtccccgaga agttgggggg aggggtcggc
     1141 aattgaaccg gtgcctagag aaggtggcgc ggggtaaact gggaaagtga tgtcgtgtac
     1201 tggctccgcc tttttcccga gggtggggga gaaccgtata taagtgcagt agtcgccgtg
     1261 aacgttcttt ttcgcaacgg gtttgccgcc agaacacagg taagtgccgt gtgtggttcc
     1321 cgcgggcctg gcctctttac gggttatggc ccttgcgtgc cttgaattac ttccacctgg
     1381 ctgcagtacg tgattcttga tcccgagctt cgggttggaa gtgggtggga gagttcgagg
     1441 ccttgcgctt aaggagcccc ttcgcctcgt gcttgagttg aggcctggcc tgggcgctgg
     1501 ggccgccgcg tgcgaatctg gtggcacctt cgcgcctgtc tcgctgcttt cgataagtct
     1561 ctagccattt aaaatttttg atgacctgct gcgacgcttt ttttctggca agatagtctt
     1621 gtaaatgcgg gccaagatct gcacactggt atttcggttt ttggggccgc gggcggcgac
     1681 ggggcccgtg cgtcccagcg cacatgttcg gcgaggcggg gcctgcgagc gcggccaccg
     1741 agaatcggac gggggtagtc tcaagctggc cggcctgctc tggtgcctgg cctcgcgccg
     1801 ccgtgtatcg ccccgccctg ggcggcaagg ctggcccggt cggcaccagt tgcgtgagcg
     1861 gaaagatggc cgcttcccgg ccctgctgca gggagctcaa aatggaggac gcggcgctcg
     1921 ggagagcggg cgggtgagtc acccacacaa aggaaaaggg cctttccgtc ctcagccgtc
     1981 gcttcatgtg actccacgga gtaccgggcg ccgtccaggc acctcgatta gttctcgagc
     2041 ttttggagta cgtcgtcttt aggttggggg gaggggtttt atgcgatgga gtttccccac
     2101 actgagtggg tggagactga agttaggcca gcttggcact tgatgtaatt ctccttggaa
     2161 tttgcccttt ttgagtttgg atcttggttc attctcaagc ctcagacagt ggttcaaagt
     2221 ttttttcttc catttcaggt gtcgtgagga attagcttgg tactaatacg actcactata
     2281 gggagacgaa ttccacaact ttgtatacaa aagttgaacg agaaacgtaa aatgatataa
     2341 atatcaatat attaaattag attttgcata aaaaacagac tacataatac tgtaaaacac
     2401 aacatatcca gtcactatga atcaactact tagatggtat tagtgacctg tactgcaggg
     2461 cggccgcgat atcccctata gtgagtcgta ttacatggtc atagctgttt cctggcagct
     2521 ctggcccgtg tctcaaaatc tctgatgtta cattgcacaa gataaaaata tatcatcatg
     2581 aacaataaaa ctgtctgctt acataaacag taatacaagg ggtgttatga gccatattca
     2641 acgggaaacg tcgaggccgc gattaaattc caacatggat gctgatttat atgggtataa
     2701 atgggctcgc gataatgtcg ggcaatcagg tgcgacaatc tatcgcttgt atgggaagcc
     2761 cgatgcgcca gagttgtttc tgaaacatgg caaaggtagc gttgccaatg atgttacaga
     2821 tgagatggtc agactaaact ggctgacgga atttatgcct cttccgacca tcaagcattt
     2881 tatccgtact cctgatgatg catggttact caccactgcg atccccggaa aaacagcatt
     2941 ccaggtatta gaagaatatc ctgattcagg tgaaaatatt gttgatgcgc tggcagtgtt
     3001 cctgcgccgg ttgcattcga ttcctgtttg taattgtcct tttaacagcg atcgcgtatt
     3061 tcgtctcgct caggcgcaat cacgaatgaa taacggtttg gttgatgcga gtgattttga
     3121 tgacgagcgt aatggctggc ctgttgaaca agtctggaaa gaaatgcata aacttttgcc
     3181 attctcaccg gattcagtcg tcactcatgg tgatttctca cttgataacc ttatttttga
     3241 cgaggggaaa ttaataggtt gtattgatgt tggacgagtc ggaatcgcag accgatacca
     3301 ggatcttgcc atcctatgga actgcctcgg tgagttttct ccttcattac agaaacggct
     3361 ttttcaaaaa tatggtattg ataatcctga tatgaataaa ttgcagtttc atttgatgct
     3421 cgatgagttt ttctaatcag aattggttaa ttggttgtaa cactggcaga gcattacgct
     3481 gacttgacgg gacggcgcaa gctcatgacc aaaatccctt aacgtgagtt acgcgtcgtt
     3541 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     3601 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     3661 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     3721 aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     3781 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     3841 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     3901 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     3961 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     4021 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     4081 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     4141 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     4201 cctggccttt tgctggcctt ttgctcacat gtt
//
