LOCUS       sequence-314450-        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     complement(78..95)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    84..291
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        298..546
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     298..318
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     469..488
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(672..771)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(801..820)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(802..820)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(825..841)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(825..841)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(943..962)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             954..1763
                     /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(1021..1040)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      1910..2498
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2399..2418
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2652..2669
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(2828..2855)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(2947..3033)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     3082..3104
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3096..3113
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3097..3113
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    3130..3225
                     /gene="mutant version of attL"
                     /label=attL3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             join(3237..3267,1..50)
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
ORIGIN
        1 catacgatgt tccagattac gcttacccat acgatgttcc agattacgct taagaattcc
       61 tagagctcgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc
      121 ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa
      181 tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg
      241 gcaggacagc aagggggagg attgggaaga gaatagcagg catgctgggg aggtaccgag
      301 ggcctatttc ccatgattcc ttcatatttg catatacgat acaaggctgt tagagagata
      361 attggaatta atttgactgt aaacacaaag atattagtac aaaatacgtg acgtagaaag
      421 taataatttc ttgggtagtt tgcagtttta aaattatgtt ttaaaatgga ctatcatatg
      481 cttaccgtaa cttgaaagta tttcgatttc ttggctttat atatcttgtg gaaaggacga
      541 aacaccgcta tcagtgatag agaacgagtt tcagagctat gctggaaaca gcatagcaag
      601 ttgaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttg
      661 cggccgcagg aacccagctt tcttgtacaa agttggcatt ataagaaagc attgcttatc
      721 aatttgttgc aacgaacagg tcactatcag tcaaaataaa atcattattt gccatccagc
      781 tgcagggcgg ccgcgatatc ccctatagtg agtcgtatta catggtcata gctgtttcct
      841 ggcagctctg gcccgtgtct caaaatctct gatgttacat tgcacaagat aaaaatatat
      901 catcatgaac aataaaactg tctgcttaca taaacagtaa tacaaggggt gttatgagcc
      961 atattcaacg ggaaacgtcg aggccgcgat taaattccaa catggatgct gatttatatg
     1021 ggtataaatg ggctcgcgat aatgtcgggc aatcaggtgc gacaatctat cgcttgtatg
     1081 ggaagcccga tgcgccagag ttgtttctga aacatggcaa aggtagcgtt gccaatgatg
     1141 ttacagatga gatggtcaga ctaaactggc tgacggaatt tatgcctctt ccgaccatca
     1201 agcattttat ccgtactcct gatgatgcat ggttactcac cactgcgatc cccggaaaaa
     1261 cagcattcca ggtattagaa gaatatcctg attcaggtga aaatattgtt gatgcgctgg
     1321 cagtgttcct gcgccggttg cattcgattc ctgtttgtaa ttgtcctttt aacagcgatc
     1381 gcgtatttcg tctcgctcag gcgcaatcac gaatgaataa cggtttggtt gatgcgagtg
     1441 attttgatga cgagcgtaat ggctggcctg ttgaacaagt ctggaaagaa atgcataaac
     1501 ttttgccatt ctcaccggat tcagtcgtca ctcatggtga tttctcactt gataacctta
     1561 tttttgacga ggggaaatta ataggttgta ttgatgttgg acgagtcgga atcgcagacc
     1621 gataccagga tcttgccatc ctatggaact gcctcggtga gttttctcct tcattacaga
     1681 aacggctttt tcaaaaatat ggtattgata atcctgatat gaataaattg cagtttcatt
     1741 tgatgctcga tgagtttttc taatcagaat tggttaattg gttgtaacac tggcagagca
     1801 ttacgctgac ttgacgggac ggcgcaagct catgaccaaa atcccttaac gtgagttacg
     1861 cgtcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     1921 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     1981 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     2041 agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     2101 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     2161 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     2221 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     2281 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     2341 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     2401 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     2461 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     2521 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg
     2581 attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa
     2641 cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc
     2701 ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga
     2761 aagcgggcag tgagcgcaac gcaattaata cgcgtaccgc tagccaggaa gagtttgtag
     2821 aaacgcaaaa aggccatccg tcaggatggc cttctgctta gtttgatgcc tggcagttta
     2881 tggcgggcgt cctgcccgcc accctccggg ccgttgcttc acaacgttca aatccgctcc
     2941 cggcggattt gtcctactca ggagagcgtt caccgacaaa caacagataa aacgaaaggc
     3001 ccagtcttcc gactgagcct ttcgttttat ttgatgcctg gcagttccct actctcgcgt
     3061 taacgctagc atggatgttt tcccagtcac gacgttgtaa aacgacggcc agtcttaagc
     3121 tcgggcccca aataatgatt ttattttgac tgatagtgac ctgttcgttg caacaaattg
     3181 atgagcaatg cttttttata atgccaactt tgtataataa agttggtggc ggatcctacc
     3241 catacgatgt tccagattac gcttacc
//
