LOCUS       sequence-425348-        4814 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Shuttling vector for an autophagy (LC3) reporter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4814)
  TITLE     Harper Lab plasmids
REFERENCE   2  (bases 1 to 4814)
  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"
COMMENT     Sequence Label: pDONR223 LC3Flux (HaloTag7-mGFP-LC3-STOP)
FEATURES             Location/Qualifiers
     source          1..4814
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(919..946)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(1038..1124)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     1173..1195
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1187..1204
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1188..1204
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    1220..1319
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     regulatory      1322..1331
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1328..2215
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="MEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSY
                     VWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIH
                     DWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIID
                     QNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVE
                     EYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLI
                     GSEIARWLSTLEISG"
     CDS             join(2237..2239,2240..2950)
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(2279..2300)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2540..2559)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2887..2908
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(3344..3443)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(3460..3479)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3461..3479)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3484..3500)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3484..3500)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             3931..4722
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
      781 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
      841 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat acgcgtaccg ctagccagga
      901 agagtttgta gaaacgcaaa aaggccatcc gtcaggatgg ccttctgctt agtttgatgc
      961 ctggcagttt atggcgggcg tcctgcccgc caccctccgg gccgttgctt cacaacgttc
     1021 aaatccgctc ccggcggatt tgtcctactc aggagagcgt tcaccgacaa acaacagata
     1081 aaacgaaagg cccagtcttc cgactgagcc tttcgtttta tttgatgcct ggcagttccc
     1141 tactctcgcg ttaacgctag catggatgtt ttcccagtca cgacgttgta aaacgacggc
     1201 cagtcttaag ctcgggcccc aaataatgat tttattttga ctgatagtga cctgttcgtt
     1261 gcaacaaatt gatgagcaat gcttttttat aatgccaact ttgtacaaaa aagcaggctc
     1321 cgccaccatg gaaatcggta ctggctttcc attcgacccc cattatgtgg aagtcctggg
     1381 cgagcgcatg cactacgtcg atgttggtcc gcgcgatggc acccctgtgc tgttcctgca
     1441 cggtaacccg acctcctcct acgtgtggcg caacatcatc ccgcatgttg caccgaccca
     1501 tcgctgcatt gctccagacc tgatcggtat gggcaaatcc gacaaaccag acctgggtta
     1561 tttcttcgac gaccacgtcc gcttcatgga tgccttcatc gaagccctgg gtctggaaga
     1621 ggtcgtcctg gtcattcacg actggggctc cgctctgggt ttccactggg ccaagcgcaa
     1681 tccagagcgc gtcaaaggta ttgcatttat ggagttcatc cgccctatcc cgacctggga
     1741 cgaatggcca gaatttgccc gcgagacctt ccaggccttc cgcaccaccg acgtcggccg
     1801 caagctgatc atcgatcaga acgtttttat cgagggtacg ctgccgatgg gtgtcgtccg
     1861 cccgctgact gaagtcgaga tggaccatta ccgcgagccg ttcctgaatc ctgttgaccg
     1921 cgagccactg tggcgcttcc caaacgagct gccaatcgcc ggtgagccag cgaacatcgt
     1981 cgcgctggtc gaagaataca tggactggct gcaccagtcc cctgtcccga agctgctgtt
     2041 ctggggcacc ccaggcgttc tgatcccacc ggccgaagcc gctcgcctgg ccaaaagcct
     2101 gcctaactgc aaggctgtgg acatcggccc aggtctgaat ctgctgcaag aagacaaccc
     2161 ggacctgatc ggcagcgaga tcgcgcgctg gctgtctact ctggagattt ccggtggttc
     2221 aggctcggga tccggggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct
     2281 ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg
     2341 cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt
     2401 gccctggccc accctcgtga ccaccctgac ctacggcgtg cagtgcttca gccgctaccc
     2461 cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga
     2521 gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga
     2581 gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa
     2641 catcctgggg cacaagctgg agtacaacta caacagccac aacgtctata tcatggccga
     2701 caagcagaag aacggcatca aggtgaactt caagatccgc cacaacatcg aggacggcag
     2761 cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct
     2821 gcccgacaac cactacctga gcacccagtc caaactgagc aaagacccca acgagaagcg
     2881 cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga
     2941 gctgtacaag ggttcaggct cgggatctgg gaattcgggt ccgtccgaga agaccttcaa
     3001 acagcgccgg agcttcgaac aaagagtgga agatgtccgg ctcatccggg agcagcaccc
     3061 caccaagatc ccagtgatta tagagcgata caagggtgag aagcagctgc ccgtcctgga
     3121 caagaccaag ttccttgtac ctgatcacgt gaatatgagc gaactcatca agataattag
     3181 aaggcgcctg cagctcaatg ctaaccaagc cttcttcctc ctggtgaatg ggcacagcat
     3241 ggtgagtgtg tccacaccca tctctgaagt gtacgagagc gagagagatg aagacggctt
     3301 cctgtacatg gtctatgcct cccaggagac gttcgggtaa tgaacccagc tttcttgtac
     3361 aaagttggca ttataagaaa gcattgctta tcaatttgtt gcaacgaaca ggtcactatc
     3421 agtcaaaata aaatcattat ttgccatcca gctgatatcc cctatagtga gtcgtattac
     3481 atggtcatag ctgtttcctg gcagctctgg cccgtgtctc aaaatctctg atgttacatt
     3541 gcacaagata aaaatatatc atcatgcctc ctctagacca gccaggacag aaatgcctcg
     3601 acttcgctgc tacccaaggt tgccgggtga cgcacaccgt ggaaacggat gaaggcacga
     3661 acccagtgga cataagcctg ttcggttcgt aagctgtaat gcaagtagcg tatgcgctca
     3721 cgcaactggt ccagaacctt gaccgaacgc agcggtggta acggcgcagt ggcggttttc
     3781 atggcttgtt atgactgttt ttttggggta cagtctatgc ctcgggcatc caagcagcaa
     3841 gcgcgttacg ccgtgggtcg atgtttgatg ttatggagca gcaacgatgt tacgcagcag
     3901 ggcagtcgcc ctaaaacaaa gttaaacatt atgagggaag cggtgatcgc cgaagtatcg
     3961 actcaactat cagaggtagt tggcgtcatc gagcgccatc tcgaaccgac gttgctggcc
     4021 gtacatttgt acggctccgc agtggatggc ggcctgaagc cacacagtga tattgatttg
     4081 ctggttacgg tgaccgtaag gcttgatgaa acaacgcggc gagctttgat caacgacctt
     4141 ttggaaactt cggcttcccc tggagagagc gagattctcc gcgctgtaga agtcaccatt
     4201 gttgtgcacg acgacatcat tccgtggcgt tatccagcta agcgcgaact gcaatttgga
     4261 gaatggcagc gcaatgacat tcttgcaggt atcttcgagc cagccacgat cgacattgat
     4321 ctggctatct tgctgacaaa agcaagagaa catagcgttg ccttggtagg tccagcggcg
     4381 gaggaactct ttgatccggt tcctgaacag gatctatttg aggcgctaaa tgaaacctta
     4441 acgctatgga actcgccgcc cgactgggct ggcgatgagc gaaatgtagt gcttacgttg
     4501 tcccgcattt ggtacagcgc agtaaccggc aaaatcgcgc cgaaggatgt cgctgccgac
     4561 tgggcaatgg agcgcctgcc ggcccagtat cagcccgtca tacttgaagc tagacaggct
     4621 tatcttggac aagaagaaga tcgcttggcc tcgcgcgcag atcagttgga agaatttgtc
     4681 cactacgtga aaggcgagat caccaaggta gtcggcaaat aaccctcgag ccacccatga
     4741 ccaaaatccc ttaacgtgag ttacgcgtcg ttccactgag cgtcagaccc cgtagaaaag
     4801 atcaaaggat cttc
//
