LOCUS       sequence-237373-        3389 bp    DNA     circular SYN 15-APR-2025
DEFINITION  pMAGIC L5-L4 entry plasmid, contains empty human U6-driven SaCas9 
            gRNA scaffold and RIP 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 3389)
  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 3389)
  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: JI702: pMAGIC (L5-L4) hU6::SaCas9 gRNA scaffol...
FEATURES             Location/Qualifiers
     source          1..3389
                     /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"
     protein_bind    complement(1504..1599)
                     /gene="mutant version of attL"
                     /label=attL4
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1630..1649)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1631..1649)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1654..1670)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1654..1670)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1772..1791)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1783..2592
                     /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(1850..1869)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      2739..3327
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3228..3247
                     /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 actctagaaa gactgagcta agaatccagc
     1081 tatcaataga aactatgaaa cagttccagg gacaaagata ccaggtcccc aacaactgca
     1141 actttctggg aaatgaggtg gaaaatgctc agccaaggaa aaagagggcc ttaccctctc
     1201 tgggacaatg attgtgctgt gaactgcttc atcaggccat ctggcccctt gttaataatc
     1261 taattaccct aggtctaagt agagttgttg acgtccaatg agcgctttct gcagacttag
     1321 cactaggcaa gtgtttggaa attacagctt cagcccctct cgccatctgc ctacctaccc
     1381 ctcctagagc ccttaatggg ccaaacggca aagtccaggg ggcagagagg aggtgctttg
     1441 gactataaag ctagtgggga cccagtaact cccaacccta agtgaccagc tacgaattcc
     1501 acccaacttt tctatacaaa gttggcatta taagaaagca ttgcttatca atttgttgca
     1561 acgaacaggt cactatcagt caaaataaaa tcattatttg ccatccagct gcagggcggc
     1621 cgcgatatcc cctatagtga gtcgtattac atggtcatag ctgtttcctg gcagctctgg
     1681 cccgtgtctc aaaatctctg atgttacatt gcacaagata aaaatatatc atcatgaaca
     1741 ataaaactgt ctgcttacat aaacagtaat acaaggggtg ttatgagcca tattcaacgg
     1801 gaaacgtcga ggccgcgatt aaattccaac atggatgctg atttatatgg gtataaatgg
     1861 gctcgcgata atgtcgggca atcaggtgcg acaatctatc gcttgtatgg gaagcccgat
     1921 gcgccagagt tgtttctgaa acatggcaaa ggtagcgttg ccaatgatgt tacagatgag
     1981 atggtcagac taaactggct gacggaattt atgcctcttc cgaccatcaa gcattttatc
     2041 cgtactcctg atgatgcatg gttactcacc actgcgatcc ccggaaaaac agcattccag
     2101 gtattagaag aatatcctga ttcaggtgaa aatattgttg atgcgctggc agtgttcctg
     2161 cgccggttgc attcgattcc tgtttgtaat tgtcctttta acagcgatcg cgtatttcgt
     2221 ctcgctcagg cgcaatcacg aatgaataac ggtttggttg atgcgagtga ttttgatgac
     2281 gagcgtaatg gctggcctgt tgaacaagtc tggaaagaaa tgcataaact tttgccattc
     2341 tcaccggatt cagtcgtcac tcatggtgat ttctcacttg ataaccttat ttttgacgag
     2401 gggaaattaa taggttgtat tgatgttgga cgagtcggaa tcgcagaccg ataccaggat
     2461 cttgccatcc tatggaactg cctcggtgag ttttctcctt cattacagaa acggcttttt
     2521 caaaaatatg gtattgataa tcctgatatg aataaattgc agtttcattt gatgctcgat
     2581 gagtttttct aatcagaatt ggttaattgg ttgtaacact ggcagagcat tacgctgact
     2641 tgacgggacg gcgcaagctc atgaccaaaa tcccttaacg tgagttacgc gtcgttccac
     2701 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     2761 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     2821 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     2881 actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     2941 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     3001 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     3061 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     3121 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     3181 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     3241 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     3301 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     3361 gccttttgct ggccttttgc tcacatgtt
//
