LOCUS       sequence-252782-        3267 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pMAGIC L3-L2 entry plasmid, contains 3xHA tag+polyA; hU6-driven 
            Sp/xCas9 TRE#2 gRNA for 3-/4-component MultiSite Gateway Pro 
            assembly. Allows C-term HA epitope fusion to dCas9 w/ TRE#2 gRNA 
            expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3267)
  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 3267)
  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: LB001: pMAGIC (L3-L2) 3x HA epitope tag + poly...
FEATURES             Location/Qualifiers
     source          1..3267
                     /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=attL3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             677..757
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     primer_bind     complement(785..802)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    791..998
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        1005..1253
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1005..1025
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1176..1195
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(1379..1478)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1508..1527)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1509..1527)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1532..1548)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1532..1548)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1650..1669)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1661..2470
                     /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(1728..1747)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      2617..3205
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3106..3125
                     /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 tgtataataa
      661 agttggtggc ggatcctacc catacgatgt tccagattac gcttacccat acgatgttcc
      721 agattacgct tacccatacg atgttccaga ttacgcttaa gaattcctag agctcgctga
      781 tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct
      841 tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca
      901 tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag
      961 ggggaggatt gggaagagaa tagcaggcat gctggggagg taccgagggc ctatttccca
     1021 tgattccttc atatttgcat atacgataca aggctgttag agagataatt ggaattaatt
     1081 tgactgtaaa cacaaagata ttagtacaaa atacgtgacg tagaaagtaa taatttcttg
     1141 ggtagtttgc agttttaaaa ttatgtttta aaatggacta tcatatgctt accgtaactt
     1201 gaaagtattt cgatttcttg gctttatata tcttgtggaa aggacgaaac accgctatca
     1261 gtgatagaga acgagtttca gagctatgct ggaaacagca tagcaagttg aaataaggct
     1321 agtccgttat caacttgaaa aagtggcacc gagtcggtgc ttttttgcgg ccgcaggaac
     1381 ccagctttct tgtacaaagt tggcattata agaaagcatt gcttatcaat ttgttgcaac
     1441 gaacaggtca ctatcagtca aaataaaatc attatttgcc atccagctgc agggcggccg
     1501 cgatatcccc tatagtgagt cgtattacat ggtcatagct gtttcctggc agctctggcc
     1561 cgtgtctcaa aatctctgat gttacattgc acaagataaa aatatatcat catgaacaat
     1621 aaaactgtct gcttacataa acagtaatac aaggggtgtt atgagccata ttcaacggga
     1681 aacgtcgagg ccgcgattaa attccaacat ggatgctgat ttatatgggt ataaatgggc
     1741 tcgcgataat gtcgggcaat caggtgcgac aatctatcgc ttgtatggga agcccgatgc
     1801 gccagagttg tttctgaaac atggcaaagg tagcgttgcc aatgatgtta cagatgagat
     1861 ggtcagacta aactggctga cggaatttat gcctcttccg accatcaagc attttatccg
     1921 tactcctgat gatgcatggt tactcaccac tgcgatcccc ggaaaaacag cattccaggt
     1981 attagaagaa tatcctgatt caggtgaaaa tattgttgat gcgctggcag tgttcctgcg
     2041 ccggttgcat tcgattcctg tttgtaattg tccttttaac agcgatcgcg tatttcgtct
     2101 cgctcaggcg caatcacgaa tgaataacgg tttggttgat gcgagtgatt ttgatgacga
     2161 gcgtaatggc tggcctgttg aacaagtctg gaaagaaatg cataaacttt tgccattctc
     2221 accggattca gtcgtcactc atggtgattt ctcacttgat aaccttattt ttgacgaggg
     2281 gaaattaata ggttgtattg atgttggacg agtcggaatc gcagaccgat accaggatct
     2341 tgccatccta tggaactgcc tcggtgagtt ttctccttca ttacagaaac ggctttttca
     2401 aaaatatggt attgataatc ctgatatgaa taaattgcag tttcatttga tgctcgatga
     2461 gtttttctaa tcagaattgg ttaattggtt gtaacactgg cagagcatta cgctgacttg
     2521 acgggacggc gcaagctcat gaccaaaatc ccttaacgtg agttacgcgt cgttccactg
     2581 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     2641 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     2701 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     2761 tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     2821 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     2881 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     2941 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     3001 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     3061 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     3121 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     3181 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     3241 cttttgctgg ccttttgctc acatgtt
//
