LOCUS       sequence-425591-        4484 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Shuttling vector for an ERphagy reporter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4484)
  TITLE     Harper Lab plasmids
REFERENCE   2  (bases 1 to 4484)
  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 ERFlux (Bip-HaloTag7-mGFP-KDEL-STOP)
FEATURES             Location/Qualifiers
     source          1..4484
                     /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     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"
     sig_peptide     1328..1381
                     /codon_start=1
                     /product="Drosophila BiP signal sequence"
                     /label=BiP signal sequence
                     /translation="MKLCILLAVVAFVGLSLG"
     CDS             1385..2269
                     /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(2276..2278,2279..2989)
                     /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(2318..2339)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2579..2598)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2926..2947
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(3014..3113)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(3130..3149)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3131..3149)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3154..3170)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3154..3170)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             3601..4392
                     /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 aagttatgca tattactggc cgtcgtggcc tttgttggcc tctcgctcgg
     1381 atccgaaatc ggtactggct ttccattcga cccccattat gtggaagtcc tgggcgagcg
     1441 catgcactac gtcgatgttg gtccgcgcga tggcacccct gtgctgttcc tgcacggtaa
     1501 cccgacctcc tcctacgtgt ggcgcaacat catcccgcat gttgcaccga cccatcgctg
     1561 cattgctcca gacctgatcg gtatgggcaa atccgacaaa ccagacctgg gttatttctt
     1621 cgacgaccac gtccgcttca tggatgcctt catcgaagcc ctgggtctgg aagaggtcgt
     1681 cctggtcatt cacgactggg gctccgctct gggtttccac tgggccaagc gcaatccaga
     1741 gcgcgtcaaa ggtattgcat ttatggagtt catccgccct atcccgacct gggacgaatg
     1801 gccagaattt gcccgcgaga ccttccaggc cttccgcacc accgacgtcg gccgcaagct
     1861 gatcatcgat cagaacgttt ttatcgaggg tacgctgccg atgggtgtcg tccgcccgct
     1921 gactgaagtc gagatggacc attaccgcga gccgttcctg aatcctgttg accgcgagcc
     1981 actgtggcgc ttcccaaacg agctgccaat cgccggtgag ccagcgaaca tcgtcgcgct
     2041 ggtcgaagaa tacatggact ggctgcacca gtcccctgtc ccgaagctgc tgttctgggg
     2101 caccccaggc gttctgatcc caccggccga agccgctcgc ctggccaaaa gcctgcctaa
     2161 ctgcaaggct gtggacatcg gcccaggtct gaatctgctg caagaagaca acccggacct
     2221 gatcggcagc gagatcgcgc gctggctgtc tactctggag atttccggtc tcgaggtgag
     2281 caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt
     2341 aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct acggcaagct
     2401 gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac
     2461 caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatga agcagcacga
     2521 cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaagga
     2581 cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg
     2641 catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga
     2701 gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga acggcatcaa
     2761 ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg ccgaccacta
     2821 ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag
     2881 cacccagtcc aaactgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga
     2941 gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaaga attcaaaaga
     3001 tgagctctga taaacccagc tttcttgtac aaagttggca ttataagaaa gcattgctta
     3061 tcaatttgtt gcaacgaaca ggtcactatc agtcaaaata aaatcattat ttgccatcca
     3121 gctgatatcc cctatagtga gtcgtattac atggtcatag ctgtttcctg gcagctctgg
     3181 cccgtgtctc aaaatctctg atgttacatt gcacaagata aaaatatatc atcatgcctc
     3241 ctctagacca gccaggacag aaatgcctcg acttcgctgc tacccaaggt tgccgggtga
     3301 cgcacaccgt ggaaacggat gaaggcacga acccagtgga cataagcctg ttcggttcgt
     3361 aagctgtaat gcaagtagcg tatgcgctca cgcaactggt ccagaacctt gaccgaacgc
     3421 agcggtggta acggcgcagt ggcggttttc atggcttgtt atgactgttt ttttggggta
     3481 cagtctatgc ctcgggcatc caagcagcaa gcgcgttacg ccgtgggtcg atgtttgatg
     3541 ttatggagca gcaacgatgt tacgcagcag ggcagtcgcc ctaaaacaaa gttaaacatt
     3601 atgagggaag cggtgatcgc cgaagtatcg actcaactat cagaggtagt tggcgtcatc
     3661 gagcgccatc tcgaaccgac gttgctggcc gtacatttgt acggctccgc agtggatggc
     3721 ggcctgaagc cacacagtga tattgatttg ctggttacgg tgaccgtaag gcttgatgaa
     3781 acaacgcggc gagctttgat caacgacctt ttggaaactt cggcttcccc tggagagagc
     3841 gagattctcc gcgctgtaga agtcaccatt gttgtgcacg acgacatcat tccgtggcgt
     3901 tatccagcta agcgcgaact gcaatttgga gaatggcagc gcaatgacat tcttgcaggt
     3961 atcttcgagc cagccacgat cgacattgat ctggctatct tgctgacaaa agcaagagaa
     4021 catagcgttg ccttggtagg tccagcggcg gaggaactct ttgatccggt tcctgaacag
     4081 gatctatttg aggcgctaaa tgaaacctta acgctatgga actcgccgcc cgactgggct
     4141 ggcgatgagc gaaatgtagt gcttacgttg tcccgcattt ggtacagcgc agtaaccggc
     4201 aaaatcgcgc cgaaggatgt cgctgccgac tgggcaatgg agcgcctgcc ggcccagtat
     4261 cagcccgtca tacttgaagc tagacaggct tatcttggac aagaagaaga tcgcttggcc
     4321 tcgcgcgcag atcagttgga agaatttgtc cactacgtga aaggcgagat caccaaggta
     4381 gtcggcaaat aaccctcgag ccacccatga ccaaaatccc ttaacgtgag ttacgcgtcg
     4441 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttc
//
