LOCUS       sequence-425587-        4661 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Shuttling vector for a mitophagy reporter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4661)
  TITLE     Harper Lab plasmids
REFERENCE   2  (bases 1 to 4661)
  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 MitoFlux (pSu9-HaloTag7-mGFP-STOP)
FEATURES             Location/Qualifiers
     source          1..4661
                     /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             1583..2467
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="EIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYV
                     WRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHD
                     WGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIIDQ
                     NVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEE
                     YMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIG
                     SEIARWLSTLEISG"
     CDS             join(2474..2476,2477..3190)
                     /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(2516..2537)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2777..2796)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3124..3145
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(3191..3290)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(3307..3326)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3308..3326)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3331..3347)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3331..3347)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             3778..4569
                     /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 gcctccactc gtgtcctcgc ctctcgcctg gcctcccaga tggctgcttc
     1381 cgccaaggtt gcccgccctg ctgtccgcgt tgctcaggtc agcaagcgca ccatccagac
     1441 tggctccccc ctccagaccc tcaagcgcac ccagatgacc tccatcgtca acgccaccac
     1501 ccgccaggct ttccagaagc gcgcctactc ttctgatcca ccggtcgcca ccggatccgg
     1561 gaattcgggc tctggttcag gcgaaatcgg tactggcttt ccattcgacc cccattatgt
     1621 ggaagtcctg ggcgagcgca tgcactacgt cgatgttggt ccgcgcgatg gcacccctgt
     1681 gctgttcctg cacggtaacc cgacctcctc ctacgtgtgg cgcaacatca tcccgcatgt
     1741 tgcaccgacc catcgctgca ttgctccaga cctgatcggt atgggcaaat ccgacaaacc
     1801 agacctgggt tatttcttcg acgaccacgt ccgcttcatg gatgccttca tcgaagccct
     1861 gggtctggaa gaggtcgtcc tggtcattca cgactggggc tccgctctgg gtttccactg
     1921 ggccaagcgc aatccagagc gcgtcaaagg tattgcattt atggagttca tccgccctat
     1981 cccgacctgg gacgaatggc cagaatttgc ccgcgagacc ttccaggcct tccgcaccac
     2041 cgacgtcggc cgcaagctga tcatcgatca gaacgttttt atcgagggta cgctgccgat
     2101 gggtgtcgtc cgcccgctga ctgaagtcga gatggaccat taccgcgagc cgttcctgaa
     2161 tcctgttgac cgcgagccac tgtggcgctt cccaaacgag ctgccaatcg ccggtgagcc
     2221 agcgaacatc gtcgcgctgg tcgaagaata catggactgg ctgcaccagt cccctgtccc
     2281 gaagctgctg ttctggggca ccccaggcgt tctgatccca ccggccgaag ccgctcgcct
     2341 ggccaaaagc ctgcctaact gcaaggctgt ggacatcggc ccaggtctga atctgctgca
     2401 agaagacaac ccggacctga tcggcagcga gatcgcgcgc tggctgtcta ctctggagat
     2461 ttccggtctc gaggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt
     2521 cgagctggac ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga
     2581 tgccacctac ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc
     2641 ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga
     2701 ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg
     2761 caccatcttc ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg
     2821 cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat
     2881 cctggggcac aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa
     2941 gcagaagaac ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt
     3001 gcagctcgcc gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc
     3061 cgacaaccac tacctgagca cccagtccaa actgagcaaa gaccccaacg agaagcgcga
     3121 tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct
     3181 gtacaagtaa acccagcttt cttgtacaaa gttggcatta taagaaagca ttgcttatca
     3241 atttgttgca acgaacaggt cactatcagt caaaataaaa tcattatttg ccatccagct
     3301 gatatcccct atagtgagtc gtattacatg gtcatagctg tttcctggca gctctggccc
     3361 gtgtctcaaa atctctgatg ttacattgca caagataaaa atatatcatc atgcctcctc
     3421 tagaccagcc aggacagaaa tgcctcgact tcgctgctac ccaaggttgc cgggtgacgc
     3481 acaccgtgga aacggatgaa ggcacgaacc cagtggacat aagcctgttc ggttcgtaag
     3541 ctgtaatgca agtagcgtat gcgctcacgc aactggtcca gaaccttgac cgaacgcagc
     3601 ggtggtaacg gcgcagtggc ggttttcatg gcttgttatg actgtttttt tggggtacag
     3661 tctatgcctc gggcatccaa gcagcaagcg cgttacgccg tgggtcgatg tttgatgtta
     3721 tggagcagca acgatgttac gcagcagggc agtcgcccta aaacaaagtt aaacattatg
     3781 agggaagcgg tgatcgccga agtatcgact caactatcag aggtagttgg cgtcatcgag
     3841 cgccatctcg aaccgacgtt gctggccgta catttgtacg gctccgcagt ggatggcggc
     3901 ctgaagccac acagtgatat tgatttgctg gttacggtga ccgtaaggct tgatgaaaca
     3961 acgcggcgag ctttgatcaa cgaccttttg gaaacttcgg cttcccctgg agagagcgag
     4021 attctccgcg ctgtagaagt caccattgtt gtgcacgacg acatcattcc gtggcgttat
     4081 ccagctaagc gcgaactgca atttggagaa tggcagcgca atgacattct tgcaggtatc
     4141 ttcgagccag ccacgatcga cattgatctg gctatcttgc tgacaaaagc aagagaacat
     4201 agcgttgcct tggtaggtcc agcggcggag gaactctttg atccggttcc tgaacaggat
     4261 ctatttgagg cgctaaatga aaccttaacg ctatggaact cgccgcccga ctgggctggc
     4321 gatgagcgaa atgtagtgct tacgttgtcc cgcatttggt acagcgcagt aaccggcaaa
     4381 atcgcgccga aggatgtcgc tgccgactgg gcaatggagc gcctgccggc ccagtatcag
     4441 cccgtcatac ttgaagctag acaggcttat cttggacaag aagaagatcg cttggcctcg
     4501 cgcgcagatc agttggaaga atttgtccac tacgtgaaag gcgagatcac caaggtagtc
     4561 ggcaaataac cctcgagcca cccatgacca aaatccctta acgtgagtta cgcgtcgttc
     4621 cactgagcgt cagaccccgt agaaaagatc aaaggatctt c
//
