LOCUS       sequence-252778-        3266 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pMAGIC L3-L2 entry plasmid, contains 3xHA tag+polyA; hU6-driven 
            Sp/xCas9 TRE#1 gRNA for 3-/4-component MultiSite Gateway Pro 
            assembly. Allows C-term HA epitope fusion to dCas9 w/ TRE#1 gRNA 
            expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3266)
  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 3266)
  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: LA901: pMAGIC (L3-L2) 3x HA epitope tag + poly...
FEATURES             Location/Qualifiers
     source          1..3266
                     /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(1378..1477)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1507..1526)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1508..1526)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1531..1547)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1531..1547)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1649..1668)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1660..2469
                     /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(1727..1746)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      2616..3204
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3105..3124
                     /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 accgtgatag
     1261 agaacgtatg tcggtttcag agctatgctg gaaacagcat agcaagttga aataaggcta
     1321 gtccgttatc aacttgaaaa agtggcaccg agtcggtgct tttttgcggc cgcaggaacc
     1381 cagctttctt gtacaaagtt ggcattataa gaaagcattg cttatcaatt tgttgcaacg
     1441 aacaggtcac tatcagtcaa aataaaatca ttatttgcca tccagctgca gggcggccgc
     1501 gatatcccct atagtgagtc gtattacatg gtcatagctg tttcctggca gctctggccc
     1561 gtgtctcaaa atctctgatg ttacattgca caagataaaa atatatcatc atgaacaata
     1621 aaactgtctg cttacataaa cagtaataca aggggtgtta tgagccatat tcaacgggaa
     1681 acgtcgaggc cgcgattaaa ttccaacatg gatgctgatt tatatgggta taaatgggct
     1741 cgcgataatg tcgggcaatc aggtgcgaca atctatcgct tgtatgggaa gcccgatgcg
     1801 ccagagttgt ttctgaaaca tggcaaaggt agcgttgcca atgatgttac agatgagatg
     1861 gtcagactaa actggctgac ggaatttatg cctcttccga ccatcaagca ttttatccgt
     1921 actcctgatg atgcatggtt actcaccact gcgatccccg gaaaaacagc attccaggta
     1981 ttagaagaat atcctgattc aggtgaaaat attgttgatg cgctggcagt gttcctgcgc
     2041 cggttgcatt cgattcctgt ttgtaattgt ccttttaaca gcgatcgcgt atttcgtctc
     2101 gctcaggcgc aatcacgaat gaataacggt ttggttgatg cgagtgattt tgatgacgag
     2161 cgtaatggct ggcctgttga acaagtctgg aaagaaatgc ataaactttt gccattctca
     2221 ccggattcag tcgtcactca tggtgatttc tcacttgata accttatttt tgacgagggg
     2281 aaattaatag gttgtattga tgttggacga gtcggaatcg cagaccgata ccaggatctt
     2341 gccatcctat ggaactgcct cggtgagttt tctccttcat tacagaaacg gctttttcaa
     2401 aaatatggta ttgataatcc tgatatgaat aaattgcagt ttcatttgat gctcgatgag
     2461 tttttctaat cagaattggt taattggttg taacactggc agagcattac gctgacttga
     2521 cgggacggcg caagctcatg accaaaatcc cttaacgtga gttacgcgtc gttccactga
     2581 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     2641 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     2701 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     2761 gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     2821 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     2881 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     2941 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     3001 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     3061 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     3121 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     3181 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     3241 ttttgctggc cttttgctca catgtt
//
