LOCUS       sequence-239026-        3772 bp    DNA     circular SYN 15-APR-2025
DEFINITION  pMAGIC L1-R5 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 3772)
  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 3772)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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: KB501: pMAGIC (L1-R5) hU6::SaCas9 gRNA scaffol...
FEATURES             Location/Qualifiers
     source          1..3772
                     /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"
     enhancer        1218..1597
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1598..1801
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1752..1772
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1798..1822
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    1835..1958
                     /gene="mutant version of attR"
                     /label=attR5
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2013..2032)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2014..2032)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2037..2053)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2037..2053)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2155..2174)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2166..2975
                     /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(2233..2252)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      3122..3710
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3611..3630
                     /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 agaacgatct atacattgaa
     1081 tcaatattgg caattagcca tattagtcat tggttatata gcataaatca atattggcta
     1141 ttggccattg catacgttgt atctatatca taatatgtac atttatattg gctcatgtcc
     1201 aatatgaccg ccatgttgac attgattatt gactagttat taatagtaat caattacggg
     1261 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc
     1321 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat
     1381 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc
     1441 ccacttggca gtacatcaag tgtatcatat gccaagtccg ccccctattg acgtcaatga
     1501 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttacgggact ttcctacttg
     1561 gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacac
     1621 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt
     1681 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaataaccc
     1741 cgccccgttg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc
     1801 tcgtttagtg aaccgtcaga tccggcgaat tccacaactt tgtatacaaa agttgaacga
     1861 gaaacgtaaa atgatataaa tatcaatata ttaaattaga ttttgcataa aaaacagact
     1921 acataatact gtaaaacaca acatatccag tcactatgaa tcaactactt agatggtatt
     1981 agtgacctgt actgcagggc ggccgcgata tcccctatag tgagtcgtat tacatggtca
     2041 tagctgtttc ctggcagctc tggcccgtgt ctcaaaatct ctgatgttac attgcacaag
     2101 ataaaaatat atcatcatga acaataaaac tgtctgctta cataaacagt aatacaaggg
     2161 gtgttatgag ccatattcaa cgggaaacgt cgaggccgcg attaaattcc aacatggatg
     2221 ctgatttata tgggtataaa tgggctcgcg ataatgtcgg gcaatcaggt gcgacaatct
     2281 atcgcttgta tgggaagccc gatgcgccag agttgtttct gaaacatggc aaaggtagcg
     2341 ttgccaatga tgttacagat gagatggtca gactaaactg gctgacggaa tttatgcctc
     2401 ttccgaccat caagcatttt atccgtactc ctgatgatgc atggttactc accactgcga
     2461 tccccggaaa aacagcattc caggtattag aagaatatcc tgattcaggt gaaaatattg
     2521 ttgatgcgct ggcagtgttc ctgcgccggt tgcattcgat tcctgtttgt aattgtcctt
     2581 ttaacagcga tcgcgtattt cgtctcgctc aggcgcaatc acgaatgaat aacggtttgg
     2641 ttgatgcgag tgattttgat gacgagcgta atggctggcc tgttgaacaa gtctggaaag
     2701 aaatgcataa acttttgcca ttctcaccgg attcagtcgt cactcatggt gatttctcac
     2761 ttgataacct tatttttgac gaggggaaat taataggttg tattgatgtt ggacgagtcg
     2821 gaatcgcaga ccgataccag gatcttgcca tcctatggaa ctgcctcggt gagttttctc
     2881 cttcattaca gaaacggctt tttcaaaaat atggtattga taatcctgat atgaataaat
     2941 tgcagtttca tttgatgctc gatgagtttt tctaatcaga attggttaat tggttgtaac
     3001 actggcagag cattacgctg acttgacggg acggcgcaag ctcatgacca aaatccctta
     3061 acgtgagtta cgcgtcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     3121 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     3181 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     3241 tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact
     3301 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     3361 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     3421 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     3481 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     3541 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     3601 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     3661 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     3721 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg tt
//
