LOCUS       sequence-425588-        5207 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Shuttling vector for a Lysophagy reporter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5207)
  TITLE     Harper Lab plasmids
REFERENCE   2  (bases 1 to 5207)
  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 LysoFlux (HaloTag7-mGFP-Galectin3-STOP)
FEATURES             Location/Qualifiers
     source          1..5207
                     /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(3737..3836)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(3853..3872)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3854..3872)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3877..3893)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3877..3893)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             4324..5115
                     /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 atggcagaca atttttcgct
     3001 ccatgatgcg ttatctgggt ctggaaaccc aaaccctcaa ggatggcctg gcgcatgggg
     3061 gaaccagcct gctggggcag ggggctaccc aggggcttcc tatcctgggg cctaccccgg
     3121 gcaggcaccc ccaggggctt atcctggaca ggcacctcca ggcgcctacc atggagcacc
     3181 tggagcttat cccggagcac ctgcacctgg agtctaccca gggccaccca gcggccctgg
     3241 ggcctaccca tcttctggac agccaagtgc ccccggagcc taccctgcca ctggccccta
     3301 tggcgcccct gctgggccac tgattgtgcc ttataacctg cctttgcctg ggggagtggt
     3361 gcctcgcatg ctgataacaa ttctgggcac ggtgaagccc aatgcaaaca gaattgcttt
     3421 agatttccaa agagggaatg atgttgcctt ccactttaac ccacgcttca atgagaacaa
     3481 caggagagtc attgtttgca atacaaagct ggataataac tggggaaggg aagaaagaca
     3541 gtcggttttc ccatttgaaa gtgggaaacc attcaaaata caagtactgg ttgaacctga
     3601 ccacttcaag gttgcagtga atgatgctca cttgttgcag tacaatcatc gggttaaaaa
     3661 actcaatgaa atcagcaaac tgggaatttc tggtgacata gacctcacca gtgcttcata
     3721 taccatgata taatgaaccc agctttcttg tacaaagttg gcattataag aaagcattgc
     3781 ttatcaattt gttgcaacga acaggtcact atcagtcaaa ataaaatcat tatttgccat
     3841 ccagctgata tcccctatag tgagtcgtat tacatggtca tagctgtttc ctggcagctc
     3901 tggcccgtgt ctcaaaatct ctgatgttac attgcacaag ataaaaatat atcatcatgc
     3961 ctcctctaga ccagccagga cagaaatgcc tcgacttcgc tgctacccaa ggttgccggg
     4021 tgacgcacac cgtggaaacg gatgaaggca cgaacccagt ggacataagc ctgttcggtt
     4081 cgtaagctgt aatgcaagta gcgtatgcgc tcacgcaact ggtccagaac cttgaccgaa
     4141 cgcagcggtg gtaacggcgc agtggcggtt ttcatggctt gttatgactg tttttttggg
     4201 gtacagtcta tgcctcgggc atccaagcag caagcgcgtt acgccgtggg tcgatgtttg
     4261 atgttatgga gcagcaacga tgttacgcag cagggcagtc gccctaaaac aaagttaaac
     4321 attatgaggg aagcggtgat cgccgaagta tcgactcaac tatcagaggt agttggcgtc
     4381 atcgagcgcc atctcgaacc gacgttgctg gccgtacatt tgtacggctc cgcagtggat
     4441 ggcggcctga agccacacag tgatattgat ttgctggtta cggtgaccgt aaggcttgat
     4501 gaaacaacgc ggcgagcttt gatcaacgac cttttggaaa cttcggcttc ccctggagag
     4561 agcgagattc tccgcgctgt agaagtcacc attgttgtgc acgacgacat cattccgtgg
     4621 cgttatccag ctaagcgcga actgcaattt ggagaatggc agcgcaatga cattcttgca
     4681 ggtatcttcg agccagccac gatcgacatt gatctggcta tcttgctgac aaaagcaaga
     4741 gaacatagcg ttgccttggt aggtccagcg gcggaggaac tctttgatcc ggttcctgaa
     4801 caggatctat ttgaggcgct aaatgaaacc ttaacgctat ggaactcgcc gcccgactgg
     4861 gctggcgatg agcgaaatgt agtgcttacg ttgtcccgca tttggtacag cgcagtaacc
     4921 ggcaaaatcg cgccgaagga tgtcgctgcc gactgggcaa tggagcgcct gccggcccag
     4981 tatcagcccg tcatacttga agctagacag gcttatcttg gacaagaaga agatcgcttg
     5041 gcctcgcgcg cagatcagtt ggaagaattt gtccactacg tgaaaggcga gatcaccaag
     5101 gtagtcggca aataaccctc gagccaccca tgaccaaaat cccttaacgt gagttacgcg
     5161 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttc
//
